Biologic preserving composition and methods of use

ABSTRACT

Disclosed herein are compositions, methods, and systems for treating skin damage or signs of aging in a subject including obtaining whole blood from the subject, collecting platelet rich plasma (PRP) from the whole blood, forming a first topical formulation having the PRP, providing the first topical formulation to the subject for application to an area of skin damage on the subject, providing a second topical formulation to the subject having an activator such that when the second topical formulation is applied to the area of skin damage on top of the first formulation, the PRP is activated and growth factors are released into the area of skin damage. Systems and kits useful in performing the methods are also disclosed.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 17/249,290, filed on Feb. 25, 2021, which is acontinuation application of U.S. patent application Ser. No. 16/818,719,filed on Mar. 13, 2020, which is a continuation-in-part of U.S. patentapplication Ser. No. 16/440,774, filed on Jun. 13, 2019, which is acontinuation-in-part of PCT Patent Application No. PCT/US2018/066895,filed Dec. 20, 2018, which claims the benefit of priority to U.S.Provisional Patent Application No. 62/610,020, filed Dec. 22, 2017, thedisclosure of each of which is incorporated by reference herein in itsentirety.

BACKGROUND Field of the Disclosure

The present disclosure relates to the topical formulations and methodsuseful for treating, preventing, or delaying skin disorders, naildisorders, hair loss, pain, and inflammation. In particular, a basecomposition is provided that maintains, prolongs, or preserves abiologic in the topical formulations. Methods of making and using thebase compositions and topical formulations are also disclosed.

DESCRIPTION

The maintenance and improvement of skin tone, elasticity, and youthfulappearance of skin is desirable by many. With age, skin loseselasticity, appears rougher, thins out, and acquires some level of skindamage. This may lead to sagging, discoloration, brown spots, UV damage,and wrinkles. Aside from surgical procedures, such as plastic surgery,to improve the look of skin or injectable treatments such as Botox® orJuvaderm®, many people resort to skin care compositions, includingprescription skin treatments, in order to maintain or improve theintegrity of their skin and prevent damage. Popular treatments includeRetin-A (tretinoin), hydroquinone, glycolic acid, and beta hydroxyl acidto remove a top layer of skin to encourage new skin growth. Manytreatments, however, that lead to improved skin tone and elasticity cantake weeks to improve the look of skin and can be very irritating. Forexample, Retin-A and hydroquinone can lead to sun sensitivity, peeling,dryness and even worsening of acne. Glycolic acid and beta hydroxy acidare not advisable for those with sensitive skin. As such, newdevelopments in skin care are desirable. Described herein arecompositions for prolonging biologics, such as PRP, for the treatment,prevention, or delay of skin disorders, nail disorders, hair loss,inflammation, and/or pain. Also described herein are methods and kitsuseful in the preparation of a formulation for such a treatment as wellas methods for treating, preventing, or delaying skin disorders, naildisorders, hair loss, or signs of aging.

SUMMARY

Treatments for skin disorders have been limited to drugs that have to betaken orally or for topical use creams and ointments. Embodimentsprovided herein relate to compositions that preserve, extend the lifeof, enhance, maintain, sustain, or otherwise prolong biologics, such ascells, proteins, antibodies, blood, serum, plasma, or componentsthereof, or other biological extracts. In some embodiments, the biologicis platelet rich plasma (PRP). In some embodiments, the compositionsthat preserve, extend the life of, enhance, maintain, sustain, orotherwise prolong biologics are referred to herein as a basecomposition.

Accordingly, some embodiments provided herein relate to basecompositions for prolonging, preserving, or maintaining a biologic. Insome embodiments, the compositions include a cell nutrient, a biologicalbuffer, a viscosity modifying agent, and a botanical extract. In someembodiments, the cell nutrient includes fetal bovine serum (FBS),glucose, human platelet lysate, bovine serum albumin, fibroblast growthsupplement, vitamins, trace elements, antioxidants, minerals, amnioticcell culture supplements, or lipopolysaccharides. In some embodiments,the biological buffer includes sodium, potassium, magnesium, calcium,alpha hydroxy acid, beta hydroxy acid, polyhydroxy acid, hyaluronicacid, carboxylic acid, or a cell culture buffering agent, or aderivative or any combination thereof, and wherein the biological bufferpreserves the biologic at about or above physiological pH. In someembodiments, the viscosity modifying agent includes polyacrylatecrosspolymer-6. In some embodiments, the botanical extract includes anaqueous ferment extract, an alcohol extract, a salicylate, a phenoliccompound, or a phytonutrient. In some embodiments, the aqueous fermentextract includes a probiotic fermented botanical extract. In someembodiments, the probiotic includes Lactobacillus, Lactobacillus casei,Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillusderivatives, Leuconostoc, Leuconostoc derivatives, Bifidobacterium,Bifidobacterium longum, Bifidobacterium derivatives, Streptococcus,Streptococcus thermophilus, Streptococcus derivatives, Saccharomycesferment filtrate, or Bacillus ferment. In some embodiments, theprobiotic fermented botanical extract includes Cocos nucifera fruitfermented with Lactobacillus, Leuconostoc kimchi, or Leuconostoc withradish root ferment filtrate. In some embodiments, the salicylateincludes an Aspen Bark isolate. In some embodiments, the phenoliccompound includes a thymol. In some embodiments, the thymol includes athymol isomer, a cresol, or O-cymen-5-ol. In some embodiments, thephytonutrient is a Sambucus nigra fruit extract derivative, Populustremuloides bark extract derivative, or Ribes nigrum fruit extractderivative.

Some embodiments provided herein relate to base compositions forprolonging, preserving, or maintaining a biologic. In some embodiments,the composition includes a cell nutrient, a biological buffer, apolymer, a thymol, and an antimicrobial. In some embodiments, thepolymer includes a natural or synthetic polymer. In some embodiments,the polymer includes a starch, a xanthan gum, a guar gum, a carrageenan,an alginate, a polysaccharide, a pectin, a gelatin, an agar, acellulose, a polyacrylate, a polyacrylamide, honey, hydrogel, chitosan,silicone, or a crosspolymer, copolymer, or derivative thereof. In someembodiments, the compositions further include a biologic, wherein thebiologic includes platelet rich plasma (PRP), fibroblast cells, adiposetissue, adipose derived stromal vascular function (SVF), nanofat,lipoaspirate components, bone marrow derived mesenchymal stem cells,adipose derived stem cells, platelet derived exosomes, adipose derivedexosomes, alpha 2 macroglobulin (A2M), human platelet lysate, orisolated microparticles thereof. In some embodiments, the biologic is alyophilized powder. In some embodiments, the base composition prolongsor enhances stability of the biologic and prolongs or enhances stabilityof components of the biologic. In some embodiments, the base compositioninhibits or prevents activation of the biologic, or wherein the basecomposition maintains, prolongs, preserves, or sustains activity of thebiologic or of already active components of the biologic. In someembodiments, the base composition is formulated for cosmetic usage,wherein the cosmetic usage includes topical application to skin.

Some embodiments provided herein relate to topical formulations. In someembodiments, the formulations include any one of the base compositionsdescribed herein, including a cell nutrient, a biological buffer, aviscosity modifying agent, and a botanical extract, and a biologic. Insome embodiments, the formulations are formulated as a cream, lotion,salve, paste, serum, gel, ointment, liquid, solution, spray, aerosol, orfoam. In some embodiments, the biologic includes platelet rich plasma(PRP), fibroblast cells, adipose tissue, adipose derived stromalvascular function (SVF), nanofat, lipoaspirate components, bone marrowderived mesenchymal stem cells, adipose derived stem cells, plateletderived exosomes, adipose derived exosomes, alpha 2 macroglobulin (A2M),human platelet lysate, or isolated microparticles thereof. In someembodiments, the topical formulation is formulated for cosmetic usage,wherein the cosmetic usage includes topical application to skin.

Some embodiments provided herein relate to methods of diminishing poresize, diminishing bacteria damage, diminishing red areas, reducing skindamage caused by sun, restoring or preventing hair loss, reducing naildisorder, or maintaining or improving skin tone in a subject in needthereof. In some embodiments, the methods include obtaining whole bloodfrom a subject having an area of skin damage, a skin disorder, a naildisorder, or hair loss, centrifuging the blood to separate platelet richplasma from platelet poor plasma and red blood cells, collecting theplatelet rich plasma (PRP), adding the base composition including a cellnutrient, a biological buffer, a viscosity modifying agent, and abotanical extract to the PRP to form PRP admixed solution therebyforming a first topical formulation, wherein the base compositionmaintains activity of the PRP for a period of time ranging from 30 to120 days, providing the first topical formulation for application to thesubject for treatment of the area of skin damage, nail damage, or hairloss, and activating the PRP. In some embodiments, activating PRPincludes increasing the temperature or applying mechanical force,wherein increasing the temperature is achieved by applying mechanicalforce to the first topical formulation or by removing the first topicalformulation from a temperature of less than about 10° C. to atemperature of at least room temperature. In some embodiments, themechanical force includes rubbing, massaging, spreading, or using amechanical device. In some embodiments, the mechanical device includes amicrodermabrasion, an oscillating fiber, a suction, a microneedling, aroller, or a mechanical cleansing device. In some embodiments, themethods further include providing a second topical formulation forapplication to the subject over the first topical formulation, whereinthe second topical formulation activates the PRP, wherein the secondtopical formulation includes a cell stimulation cocktail includingcalcium chloride, hydrochloric acid, thrombin, autologous thrombin,bovine thrombin, collagen, calcium, magnesium, sodium, components ofsnake venom, batroxobin, or combinations thereof. In some embodiments,the subject suffers from skin damage and signs of aging caused bysmoking, alcohol, diet, extreme temperatures, chemicals, stress, lack ofsleep, poor diet, poor immune system or a combination thereof. In someembodiments, the skin disorder is selected from a group consisting ofstretch marks (striae), psoriasis, skin cancer, acne, alopecia,carbuncles, dermatitis, eczema, atopic dermatitis, contact dermatitis,seborrheic dermatitis, cradle cap, perioral dermatitis, shingles,ringworm, melisma, impetigo, acne aestivalis (Mallorca acne), acneconglobate, acne cosmetica (cosmetic acne), acne fulminans (acutefebrile ulcerative acne), acne keloidalis nuchae (acne keloidalis,dermatitis papillaris capillitii, folliculitis keloidalis, folliculitiskeloidis nuchae, nuchal keloid acne), adult forehead with scattered redpimples, acne vulgaris, acne mechanica, acne medicamentosa, acnemiliaris necrotica (acne varioliformis), acne vulgaris, acne with facialedema (solid facial edema), blepharophyma, erythrotelangiectatic rosacea(erythematotelangiectatic rosacea, vascular rosacea), excoriated acne(acne excoriee des jeunes filles, Picker's acne), glandular rosacea,gnathophyma, gram-negative rosacea, granulomatous facial dermatitis,rhinophyma, granulomatous perioral dermatitis, halogen acne,hidradenitis suppurativa (acne inversa, pyoderma fistulans significa,Verneuil's disease), idiopathic facial aseptic granuloma, infantileacne, lupoid rosacea (granulomatous rosacea, micropapular tuberculid,rosacea-like tuberculid of Lewandowsky), lupus miliaris disseminatusfaciei, metophyma, neonatal acne (acne infantum, acne neonatorum,neonatal cephalic pustulosis), occupational acne, oil acne, ocularrosacea (ophthalmic rosacea, ophthalmorosacea), otophyma, periorificialdermatitis, persistent edema of rosacea (chronic upper facialerythematous edema, Morbihan's disease, rosaceous lymphedema), phymatousrosacea, pomade acne, papulopustular rosacea (inflammatory rosacea),perifolliculitis capitis abscedens et suffodiens (dissecting cellulitisof the scalp, dissecting folliculitis, perifolliculitis capitisabscedens et suffodiens of Hoffman), perioral dermatitis, periorbitaldermatitis (periocular dermatitis), pyoderma faciale (rosaceafulminans), rhinophyma, rosacea (acne rosacea), rosacea conglobate,synovitis-acne-pustulosis-hyperostosis-osteomyelitis syndrome (SAPHOsyndrome), steroid rosacea, tar acne, skin cancer, tropical acne or acombination thereof.

Accordingly, embodiments provided herein relate to methods, systems, andcompositions for preserving, extending the life of, enhancing,maintaining, sustaining, or otherwise prolonging a biologic, orcomponents of a biologic. In some embodiments, the methods includecontacting a biologic with a base composition, wherein the basecomposition is capable of preserving, extending the life of, enhancing,maintaining, sustaining, or otherwise prolonging the biologic orcomponents of a biologic. In some embodiments, the base compositionpreserves, extends the life of, enhances, maintains, sustains, orotherwise prolongs a biologic and/or components thereof for a period ofmore than 30, 40, 50, 60, 70, 80, 90, 100, or 120 days.

In some embodiments, the base compositions include a humectant, sodiumbicarbonate, a buffering agent, magnesium chloride, and a viscositymodifying agent. In some embodiments, the base compositions furtherinclude a cell culture reagent, and wherein the viscosity modifyingagent is potassium chloride, sodium chloride, or both. In someembodiments, the buffering agent is sodium citrate, sodium acetate, orboth. In some embodiments, the cell culture reagent is fetal bovineserum (FBS) or a growth medium. In some embodiments, the humectant isglucose. In some embodiments, the composition further includes sodiumphosphate. In some embodiments, the base compositions further include anemulsion stabilizer, a chelating agent, an antioxidant, an activationinhibitor, a protein stabilizer, or an enzyme inhibitor. In someembodiments, the base compositions further include a sun protectionagent, a skin conditioning agent, a biocide, a biologic buffering agent,a pH adjuster, a cell culture reagent, a skin permeability enhancer, abotanical agent, or a skin delivery system.

In some embodiments, the composition includes water, glycerin,niacinamide, polyacrylate crosspolymer-6, Lonicera caprifolium(honeysuckle) flower extract, Persea gratissima (avocado) oil, Aloebarbadensis leaf juice, sodium gluconate, sodium hyaluronate (L),Althaea officinalis (marshmallow) root extract, O-cymen-5-OL, sodiumhydroxide, glucose (D), sodium chloride, sodium citrate, sodium acetate,sodium bicarbonate, tocopheryl acetate (D-alpha), potassium phosphate,potassium chloride, magnesium chloride, phenoxyethanol, Anthemis nobilis(chamomile) flower oil, Rosmarinus officinalis (rosemary) leaf oil,Thymus vulgaris (thyme) flower/leaf oil, benzyl alcohol, caprylylglycol, caprylhydroxamic acid, or combinations thereof.

In some embodiments, the composition includes water in an amount rangingfrom about 85 to 95 wt %, polyacrylate crosspolymer-6 in an amountranging from about 0.05 to 5 wt %, Lonicera japonica (honeysuckle)flower extract is present in an amount ranging from about 0.05 to 5 wt%, glycerin in an amount ranging from about 0.05 to 5 wt %, niacinamidein an amount ranging from about 0.05 to 5 wt %, Lonicera caprifolium(honeysuckle) flower extract in an amount ranging from about 0.05 to 5wt %, Persea gratissima (avocado) oil in an amount ranging from about0.05 to 5 wt %, Aloe barbadensis leaf juice in an amount ranging fromabout 0.05 to 5 wt %, citric acid in an amount ranging from about 0.05to 5 wt %, sodium gluconate in an amount ranging from about 0.05 to 5 wt%, sodium hyaluronate (L) in an amount ranging from about 0.01 to 5 wt%, Althaea officinalis (marshmallow) root extract in an amount rangingfrom about 0.01 to 5 wt %, O-cymen-5-OL in an amount ranging from about0.01 to 5 wt %, glucose (D) in an amount ranging from about 0.01 to 5 wt%, sodium chloride in an amount ranging from about 0.001 to 5 wt %,sodium citrate in an amount ranging from about 0.001 to 5 wt %, sodiumacetate in an amount ranging from about 0.001 to 5 wt %, sodiumbicarbonate in an amount ranging from about 0.001 to 5 wt %, tocopherylacetate (D-alpha) in an amount ranging from about 0.001 to 5 wt %,potassium phosphate in an amount ranging from about 0.001 to 5 wt %,potassium chloride in an amount ranging from about 0.001 to 5 wt %,and/or magnesium chloride in an amount ranging from about 0.001 to 5 wt%.

In some embodiments, the water is present in an amount of about 92.72 wt%, polyacrylate crosspolymer-6 is present in an amount of about 1.3 wt%, Lonicera japonica (honeysuckle) flower extract is present in anamount of about 1.2 wt %, glycerin is present in an amount of about 1.2wt %, niacinamide is present in an amount of about 1 wt %, Loniceracaprifolium (honeysuckle) flower extract is present in an amount ofabout 0.5%, Persea gratissima (avocado) oil is present in an amount ofabout 0.5 wt %, Aloe barbadensis leaf juice is present in an amount ofabout 0.5 wt %, citric acid is present in an amount of about 0.3 wt %,sodium gluconate is present in an amount of about 0.2 wt %, sodiumhyaluronate (L) is present in an amount of about 0.2 wt %, Althaeaofficinalis (marshmallow) root extract is present in an amount of about0.1 wt %, O-cymen-5-OL is present in an amount of about 0.1 wt %,glucose (D) is present in an amount of about 0.1 wt %, sodium chlorideis present in an amount of about 0.01 wt %, sodium citrate is present inan amount of about 0.01 wt %, sodium acetate is present in an amount ofabout 0.01 wt %, sodium bicarbonate is present in an amount of about0.01 wt %, tocopheryl acetate (D-alpha) is present in an amount of about0.01 wt %, potassium phosphate is present in an amount of about 0.01 wt%, potassium chloride is present in an amount of about 0.01 wt %, and/ormagnesium chloride is present in an amount of about 0.01 wt %.

In some embodiments, the base compositions further include a biologic,wherein the biologic is platelet rich plasma (PRP), fibroblast cells,adipose tissue, adipose derived stromal vascular function (SVF),nanofat, lipoaspirate components, bone marrow derived mesenchymal stemcells, adipose derived stem cells, platelet derived exosomes, adiposederived exosomes, alpha 2 macroglobulin (A2M), human platelet lysate, orisolated microparticles thereof. In some embodiments, the basecompositions further include PRP and a second biologic, wherein thesecond biologic is fibroblast cells, adipose tissue, adipose derivedstromal vascular function (SVF), nanofat, lipoaspirate components, bonemarrow derived mesenchymal stem cells, adipose derived stem cells,platelet derived exosomes, adipose derived exosomes, alpha 2macroglobulin (A2M), human platelet lysate, or isolated microparticlesthereof. In some embodiments, the biologic or the second biologic is alyophilized powder. In some embodiments, the base composition prolongsor enhances stability of the biologic and prolongs or enhances stabilityof components of the biologic. In some embodiments, the biologic or thesecond biologic is PRP, and wherein the components of the biologic orthe second biologic includes growth factors, proteins, or nucleic acids.In some embodiments, the base composition inhibits or preventsactivation of the biologic or the second biologic, or wherein the basecomposition maintains, prolongs, preserves, or sustains activity of thebiologic or the second biologic or of already active components of thebiologic or the second biologic.

Some embodiments provided herein relate to a topical formulation thatincludes a base composition according to any of the base compositionsdescribed herein and a biologic. In some embodiments, the biologic isplatelet rich plasma (PRP), fibroblast cells, adipose tissue, adiposederived stromal vascular function (SVF), nanofat, lipoaspiratecomponents, bone marrow derived mesenchymal stem cells, adipose derivedstem cells, platelet derived exosomes, adipose derived exosomes, alpha 2macroglobulin (A2M), human platelet lysate, or isolated microparticlesthereof. In some embodiments, the formulation is formulated as a cream,lotion, salve, paste, serum, gel, ointment, liquid, solution, spray,aerosol, or foam.

Some embodiments provided herein relate to methods of making a topicalformulation. In some embodiments, the methods include mixing a biologicwith a base composition according to any of the base compositionsdescribed herein. In some embodiments, the base composition maintainsactivity of the biologic for a period of 30, 45, 60, 75, 90 105, or 120days, or longer. In some embodiments, the biologic is a serum or apowder. In some embodiments, the biologic is a lyophilized powder, andwherein the biologic powder is stable for at least 24 months. In someembodiments, the formulation is formulated for topical application to anarea of skin having skin damage, and wherein the formulation improvesappearance of the skin. In some embodiments, the formulation isformulated for use in combination with a skin obstruction device. Insome embodiments, the skin obstruction device includes electroporation,radiofrequency, ultrasound, high intensity focused ultrasound, needling,intense pulsed light (IPL), ablative laser, non-ablative laser,microdermabrasion, hydradermabrasion, iontophoresis, chemical peel,plasma, high velocity air, high velocity aqueous solution, orcombinations thereof. In some embodiments, the biologic includesplatelet rich plasma (PRP), fibroblast cells, adipose tissue, adiposederived stromal vascular function (SVF), nanofat, lipoaspiratecomponents, bone marrow derived mesenchymal stem cells, adipose derivedstem cells, platelet derived exosomes, adipose derived exosomes, alpha 2macroglobulin (A2M), human platelet lysate, or isolated microparticlesthereof, or combinations thereof.

Some embodiments provided herein relate to a method of diminishing poresize, diminishing bacteria damage, diminishing red areas, reducing skindamage caused by sun, and maintaining or improving skin tone in asubject in need thereof. In some embodiments the methods includeobtaining whole blood from a subject having an area of skin damage,centrifuging the blood to separate platelet rich plasma from plateletpoor plasma and red blood cells, collecting the platelet rich plasma(PRP), adding a base composition according to any of the basecompositions provided herein to the PRP to form PRP admixed solutionthereby forming a first topical formulation, providing the first topicalformulation for application to the subject for treatment of the area ofskin damage, and providing a second topical formulation for applicationto the subject over the area of skin damage that has received the firsttopical formulation. In some embodiments, the second topical formulationactivates the platelet rich plasma. In some embodiments, the basecomposition maintains activity of the PRP for a period of time rangingfrom at least 30 to at least 120 days. In some embodiments, the secondformulation is a gel, liquid, cream, foam, or lotion. In someembodiments, the second formulation is a liquid and wherein the liquidis applied as a spray such as from an aerosol spray or pump spray. Insome embodiments, the second formulation includes a platelet rich plasmaactivator. In some embodiments, the second formulation includes calciumchloride, thrombin, autologous thrombin, bovine thrombin, collagen,calcium, magnesium, sodium, components of snake venom, batroxobin, orcombinations thereof. In some embodiments, the subject suffers from skindamage and signs of aging caused by smoking, alcohol, diet, extremetemperatures, chemicals, stress, lack of sleep, poor diet, poor immunesystem or a combination thereof. In some embodiments, the subjectsuffers from a skin disorder, a nail disorder, or hair loss. In someembodiments, the skin disorder causes skin damage and signs of aging. Insome embodiments, the skin disorder is selected from a group consistingof stretch marks (striae), psoriasis, skin cancer, acne, alopecia,carbuncles, dermatitis, eczema, atopic dermatitis, contact dermatitis,seborrheic dermatitis, cradle cap, perioral dermatitis, shingles,ringworm, melisma, impetigo, acne aestivalis (Mallorca acne), acneconglobate, acne cosmetica (cosmetic acne), acne fulminans (acutefebrile ulcerative acne), acne keloidalis nuchae (acne keloidalis,dermatitis papillaris capillitii, folliculitis keloidalis, folliculitiskeloidis nuchae, nuchal keloid acne), adult forehead with scattered redpimples, acne vulgaris, acne mechanica, acne medicamentosa, acnemiliaris necrotica (acne varioliformis), acne vulgaris, acne with facialedema (solid facial edema), blepharophyma, erythrotelangiectatic rosacea(erythematotelangiectatic rosacea, vascular rosacea), excoriated acne(acne excoriee des jeunes filles, Picker's acne), glandular rosacea,gnathophyma, gram-negative rosacea, granulomatous facial dermatitis,rhinophyma, granulomatous perioral dermatitis, halogen acne,hidradenitis suppurativa (acne inversa, pyoderma fistulans significa,Verneuil's disease), idiopathic facial aseptic granuloma, infantileacne, lupoid rosacea (granulomatous rosacea, micropapular tuberculid,rosacea-like tuberculid of Lewandowsky), lupus miliaris disseminatusfaciei, metophyma, neonatal acne (acne infantum, acne neonatorum,neonatal cephalic pustulosis), occupational acne, oil acne, ocularrosacea (ophthalmic rosacea, ophthalmorosacea), otophyma, periorificialdermatitis, persistent edema of rosacea (chronic upper facialerythematous edema, Morbihan's disease, rosaceous lymphedema), phymatousrosacea, pomade acne, papulopustular rosacea (inflammatory rosacea),perifolliculitis capitis abscedens et suffodiens (dissecting cellulitisof the scalp, dissecting folliculitis, perifolliculitis capitisabscedens et suffodiens of Hoffman), perioral dermatitis, periorbitaldermatitis (periocular dermatitis), pyoderma faciale (rosaceafulminans), rhinophyma, rosacea (acne rosacea), rosacea conglobate,synovitis-acne-pustulosis-hyperostosis-osteomyelitis syndrome (SAPHOsyndrome), steroid rosacea, tar acne, skin cancer, tropical acne or acombination thereof. In some embodiments, the subject has wrinkles, acnescars, reduced skin elasticity, sagging skin, increased skin dryness,rashes, redness, translucency, fine lines, loss of radiance, increase inthe dullness of skin, uneven pigmentation, discoloration, blotchiness,scarring, rough and leathery appearance, freckles, moles, actinickeratosis, slower wound healing, easy bruising and tearing, ruddiness,uneven texture, fine lines, age spots, or a combination thereof. In someembodiments, treating, preventing, or delaying hair loss includes hairrestoration or maintenance of hair health. In some embodiments, the basecomposition is a serum, lotion, liquid primer, cream, gel or acombination thereof. In some embodiments, the base composition furtherincludes at least one keratolytic agent, at least one anti-inflammatoryagent, sun protection agent, preservative for platelets in the plateletrich plasma, nutrient, at least one botanical or a combination thereof.In some embodiments, the activating step further includes degranulatingplatelets in the platelet rich plasma of the first topical formulationand releasing growth factors from the platelets into and through theskin of the subject. In some embodiments, the method further includesperforming skin obstruction of the skin over the area of skin damage. Insome embodiments, the skin obstruction includes electroporation,radiofrequency, ultrasound, high intensity focused ultrasound, needling,intense pulsed light (IPL), ablative laser, non-ablative laser,microdermabrasion, hydradermabrasion, iontophoresis, chemical peel,plasma, high velocity air, high velocity aqueous solution, orcombinations thereof.

Some embodiments provided herein relate to a kit for preparing a basecomposition. In some embodiments, the kit includes a first vesselincluding lyophilized biologic powder and a second vessel containing abase composition according to any base compositions described herein. Insome embodiments, the kit further includes a dissolution solution fordissolving or solubilizing the lyophilized biologic powder in the firstvessel. In some embodiments, the biologic is platelet rich plasma (PRP),fibroblast cells, adipose tissue, adipose derived stromal vascularfunction (SVF), nanofat, lipoaspirate components, bone marrow derivedmesenchymal stem cells, adipose derived stem cells, platelet derivedexosomes, adipose derived exosomes, alpha 2 macroglobulin (A2M), humanplatelet lysate, or isolated microparticles thereof, or combinationsthereof. In some embodiments, the dissolution solution includes water,an aqueous solution, saline, sodium chloride, or phosphate bufferedsaline. In some embodiments, the kit further includes an activatorformulation. In some embodiments, the activator formulation includescalcium chloride, thrombin, autologous thrombin, bovine thrombin,collagen, calcium, magnesium, sodium, components of snake venom,batroxobin, or combinations thereof. In some embodiments, the activatorformulation is formulated as a spray.

In some embodiments, a first formulation is applied to or administeredto a subject, for example by topical application. Thus, in someembodiments, the first formulation including a base composition and abiologic is formulated as a topical cosmetic base, a gel, a lotion, anointment, a spray, an aerosol, a powder, a solution, a liquid, a foam, asalve, a paste, a serum, or a cream or combination thereof. In someembodiments, the first formulation is applied by spraying, rubbing,massaging, spreading, coating, or otherwise topically applying to asubject.

Some embodiments provided herein relate to applying a second formulationto the subject following application of the first formulation, therebyactivating the first formulation, or activating the biologic in thefirst formulation, such as by activating the PRP of the firstformulation. In some embodiments, the second formulation includes anactivator, such as, for example, calcium chloride, autologous thrombin,batroxobin, bovine thrombin, collagen, or other activator of thespecific biologic within the first formulation.

Some embodiments provided herein relate to the use of a biologic, suchas PRP, taken from a subject suffering from a disorder, such as a skindisorder, nail disorder, or hair loss. These cells can be activated andfurther formulations may be administered to ensure the survival of thecells. Activation can also lead to degranulation, allowing growthfactors to be released into and through the skin to enhance the healingof a skin disorder or nail disorder, for hair restoration ormaintenance, or for preventing signs of aging in skin. In someembodiments, the biologic, such as PRP, is taken from a subject that isnot suffering from a disorder, and is used to prevent or delay the onsetof a disorder, such as a skin disorder, nail disorder, or hair loss.

Some embodiments provided herein relate to methods of treating skin on asubject suffering from skin damage and/or signs of aging is provided.The method includes obtaining whole blood from a subject having skindamage, centrifuging the blood to separate platelet rich plasma fromplatelet poor plasma and red blood cells, collecting the platelet richplasma (PRP), adding a base composition, wherein the base compositionpreserves, extends the life of, enhances, maintains, sustains, orotherwise prolongs the PRP for a period of more than 30, 40, 50, 60, 70,80, 90, 100, or 120 days. In some embodiments, the base composition is atopical cosmetic base, a gel, a serum, a lotion, an ointment, a spray,an aerosol, a powder, a solution, a liquid, a foam, a salve, a paste, ora cream or combination thereof. In some embodiments, the basecomposition is added to the platelet rich plasma to form platelet richplasma admixed solution thereby forming a first topical formulation. Insome embodiments, the method includes providing the first topicalformulation to the subject for treatment of the skin damage for a 90 daytime period. In some embodiments, the PRP is preserved, extended,enhanced, maintained, sustained, or otherwise prolonged due to the basecomposition for a period of more than 30, 40, 50, 60, 70, 80, 90, 100,or 120 days, such that the first topical formulation may be used havingfunctional PRP at a time period of more than 30, 40, 50, 60, 70, 80, 90,100, or 120 days after being prepared.

In some embodiments, the providing includes instructions for the subjectto topically apply the first topical formulation to an area of the skinand skin damage 1, 2, 3 or 4 times a day, for up to 30, 60, 90 or 120days or any number of days in between a range defined by any twoaforementioned values, and activating the platelet rich plasma. In someembodiments, the activating includes providing a second topicalformulation to the subject. In some embodiments, the providing includesapplying the second topical formulation over the area of the skin andskin damage that has received the first topical formulation. In someembodiments, treating the skin damage and signs of aging is selectedfrom diminishing pore size, diminishing bacteria damage, diminishing redareas, reducing skin damage caused by sun, and maintaining or improvingskin tone.

In some embodiments, the second formulation is a gel, liquid, cream orlotion. In some embodiments, the second formulation is a liquid andwherein the liquid is applied as a spray such as an aerosol spray orpump spray. In some embodiments, the second formulation includes aplatelet rich plasma activator. In some embodiments, the secondformulation includes calcium, magnesium, sodium, components of snakevenom or combinations thereof. In some embodiments, the component ofsnake venom includes Batroxobin. The calcium may be provided as calciumchloride or calcium carbonate, for example. In some embodiments, thesecond formulation further includes Batroxobin, epinephrine and/orthrombin. In some embodiments, wherein the second formulation isprovided as a spray, the second formulation includes Aqua (Water), aplatelet activator, Polysorbate 80, Propylene Glycol, Sodium Chloride,Anthemis nobilis (Chamomile) Flower Oil, Rosa canina (Rose Hip) FruitOil, Thymus vulgaris (Thyme) Flower/Leaf Oil, Althaea officinalis(Marshmallow) Root Extract, Hamamelis virginiana (Witch Hazel) Water,Alcohol, Glycerin, Ethylhexylglycerin, Citric Acid, Potassium Sorbate,Phenoxyethanol, or combinations thereof. In some embodiments, theplatelet activator is calcium chloride.

In some embodiments, the subject is also suffering from a skin disorder.In some embodiments, the skin damage and signs of aging are caused bythe skin disorder. In some embodiments, the skin damage and signs ofaging are caused by smoking, alcohol, diet, extreme temperatures,chemicals, stress, lack of sleep, poor diet, poor immune system or acombination thereof.

In some embodiments, the skin disorder is selected from a groupconsisting of stretch marks (striae), psoriasis, skin cancer, acne,alopecia, carbuncles, dermatitis, eczema, atopic dermatitis, contactdermatitis, seborrheic dermatitis, cradle cap, perioral dermatitis,shingles, ringworm, melisma, impetigo, acne aestivalis (Mallorca acne),acne conglobate, acne cosmetica (cosmetic acne), acne fulminans (acutefebrile ulcerative acne), acne keloidalis nuchae (acne keloidalis,dermatitis papillaris capillitii, folliculitis keloidalis, folliculitiskeloidis nuchae, nuchal keloid acne), adult forehead with scattered redpimples, acne vulgaris, acne mechanica, acne medicamentosa, acnemiliaris necrotica (acne varioliformis), acne vulgaris, acne with facialedema (solid facial edema), blepharophyma, erythrotelangiectatic rosacea(erythematotelangiectatic rosacea, vascular rosacea), excoriated acne(acne excoriee des jeunes filles, Picker's acne), glandular rosacea,gnathophyma, gram-negative rosacea, granulomatous facial dermatitis,rhinophyma, granulomatous perioral dermatitis, halogen acne,hidradenitis suppurativa (acne inversa, pyoderma fistulans significa,Verneuil's disease), idiopathic facial aseptic granuloma, infantileacne, lupoid rosacea (granulomatous rosacea, micropapular tuberculid,rosacea-like tuberculid of Lewandowsky), lupus miliaris disseminatusfaciei, metophyma, neonatal acne (acne infantum, acne neonatorum,neonatal cephalic pustulosis), occupational acne, oil acne, ocularrosacea (ophthalmic rosacea, ophthalmorosacea), otophyma, periorificialdermatitis, persistent edema of rosacea (chronic upper facialerythematous edema, Morbihan's disease, rosaceous lymphedema), phymatousrosacea, pomade acne, papulopustular rosacea (inflammatory rosacea),perifolliculitis capitis abscedens et suffodiens (dissecting cellulitisof the scalp, dissecting folliculitis, perifolliculitis capitisabscedens et suffodiens of Hoffman), perioral dermatitis, periorbitaldermatitis (periocular dermatitis), pyoderma faciale (rosaceafulminans), rhinophyma, rosacea (acne rosacea), rosacea conglobate,synovitis-acne-pustulosis-hyperostosis-osteomyelitis syndrome (SAPHOsyndrome), steroid rosacea, tar acne, skin cancer, tropical acne or acombination thereof.

In some embodiments, the subject has wrinkles, acne scars, reduced skinelasticity, sagging skin, increased skin dryness, rashes, redness,translucency, fine lines, loss of radiance, increase in the dullness ofskin, uneven pigmentation, discoloration, blotchiness, scarring, roughand leathery appearance, freckles, moles, actinic keratosis, slowerwound healing, easy bruising and tearing, ruddiness, uneven texture,fine lines, age spots, or a combination thereof.

In some embodiments, the subject is suffering from hair loss or hairdamage. In some embodiments, the subject is not suffering from hair lossor hair damage. The compositions described herein may be used forpreventing hair loss, hair restoration, the slowing of hair loss, or formaintenance of hair health. The compositions provided herein may beprovided to an area of hair loss, or to slow hair loss, or to the hairfor hair maintenance in the form of a salve, a spray, a shampoo, aconditioner, a hair follicle spray, a serum, an injectable composition,or any other formulations suitable for application to hair.

In some embodiments, the topical cosmetic base is a gel, a serum, alotion, an ointment, a spray, an aerosol, a powder, a solution, aliquid, a foam, a salve, a paste, or a cream, or a combination thereof.In some embodiments, the topical cosmetic base further includes at leastone keratolytic agent, at least one anti-inflammatory agent, sunprotection agent, preservative for platelets in the platelet richplasma, nutrient or a combination thereof.

In some embodiments, the activating step further includes degranulatingplatelets in the platelet rich plasma of the first topical formulationand releasing growth factors from the platelets into and through theskin of the subject.

In a second aspect, a method of treating a subject suffering from skindamage and signs of aging is provided. The method includes obtainingfibroblast skin cells from skin of a subject, placing the fibroblastskin cells in growth media, growing the fibroblast skin cells up toconfluency; mixing the fibroblast cells with a topical cosmetic base,thereby making a first formulation including fibroblast cells andapplying the formulation to the skin of the subject.

In some embodiments, the subject is also suffering from a skin disorder.In some embodiments, the skin damage and signs of aging are caused bythe skin disorder. In some embodiments, the skin damage and signs ofaging are caused by smoking, alcohol, diet, extreme temperatures,chemicals, stress, lack of sleep, poor diet, poor immune system or acombination thereof. In some embodiments, the skin disorder is selectedfrom a group consisting of stretch marks (striae), psoriasis, skincancer, acne, alopecia, carbuncles, dermatitis, eczema, atopicdermatitis, contact dermatitis, seborrheic dermatitis, cradle cap,perioral dermatitis, shingles, ringworm, melisma, impetigo, acneaestivalis (Mallorca acne), acne conglobate, acne cosmetica (cosmeticacne), acne fulminans (acute febrile ulcerative acne), acne keloidalisnuchae (acne keloidalis, dermatitis papillaris capillitii, folliculitiskeloidalis, folliculitis keloidis nuchae, nuchal keloid acne), adultforehead with scattered red pimples, acne vulgaris, acne mechanica, acnemedicamentosa, acne miliaris necrotica (acne varioliformis), acnevulgaris, acne with facial edema (solid facial edema), blepharophyma,erythrotelangiectatic rosacea (erythematotelangiectatic rosacea,vascular rosacea), excoriated acne (acne excoriee des jeunes filles,Picker's acne), glandular rosacea, gnathophyma, gram-negative rosacea,granulomatous facial dermatitis, rhinophyma, granulomatous perioraldermatitis, halogen acne, hidradenitis suppurativa (acne inversa,pyoderma fistulans significa, Verneuil's disease), idiopathic facialaseptic granuloma, infantile acne, lupoid rosacea (granulomatousrosacea, micropapular tuberculid, rosacea-like tuberculid ofLewandowsky), lupus miliaris disseminatus faciei, metophyma, neonatalacne (acne infantum, acne neonatorum, neonatal cephalic pustulosis),occupational acne, oil acne, ocular rosacea (ophthalmic rosacea,ophthalmorosacea), otophyma, periorificial dermatitis, persistent edemaof rosacea (chronic upper facial erythematous edema, Morbihan's disease,rosaceous lymphedema), phymatous rosacea, pomade acne, papulopustularrosacea (inflammatory rosacea), perifolliculitis capitis abscedens etsuffodiens (dissecting cellulitis of the scalp, dissecting folliculitis,perifolliculitis capitis abscedens et suffodiens of Hoffman), perioraldermatitis, periorbital dermatitis (periocular dermatitis), pyodermafaciale (rosacea fulminans), rhinophyma, rosacea (acne rosacea), rosaceaconglobate, synovitis-acne-pustulosis-hyperostosis-osteomyelitissyndrome (SAPHO syndrome), steroid rosacea, tar acne, skin cancer,tropical acne or a combination thereof.

In some embodiments, the subject has wrinkles, acne scars, reduced skinelasticity, sagging skin, increased skin dryness, rashes, redness,translucency, fine lines, loss of radiance, increase in the dullness ofskin, uneven pigmentation, discoloration, blotchiness, scarring, roughand leathery appearance, freckles, moles, actinic keratosis, slowerwound healing, easy bruising and tearing, ruddiness, uneven texture,fine lines, age spots, or a combination thereof.

In some embodiments, the topical cosmetic base is a serum, lotion,liquid primer, cream, gel, or liquid or a combination thereof. In someembodiments, the topical cosmetic base further includes at least onekeratolytic agent, at least one anti-inflammatory agent, sun protectionagent, or a combination thereof.

In some embodiments, the fibroblast cells are obtained from the skin onthe neck, arms, legs, buttocks, stomach, back or behind the ear of thesubject.

In some embodiments, the method further includes freezing the fibroblastcells and storing the fibroblast cells prior to mixing fibroblast cellswith the topical cosmetic base.

In some embodiments, the method further including thawing the fibroblastcells prior to mixing the fibroblast cells with the topical cosmeticbase.

In some embodiments, the growth media includes human platelet lysate,platelet rich plasma, human serum or a combination thereof

In some embodiments, wherein growth media includes human platelet lysateand wherein the human platelet lysate is from the subject, the humanplatelet lysate is obtained by obtaining blood from the subject,centrifuging the blood to separate platelet-poor plasma, platelet richplasma and collecting the platelet rich plasma.

In some embodiments, the method further includes subjecting the plateletrich plasma to freeze thaw cycles, thereby forming human plateletlysate.

In some embodiments, the method further includes adding and mixing theplatelet rich plasma, human platelet lysate or a combination thereof,with growth media, prior to placing the fibroblast skin cells in thegrowth media.

In some embodiments, the method further includes activating platelets inthe platelet rich plasma. The method includes providing a secondformulation to the subject, wherein the providing includes applying thesecond topical formulation over the area of the skin and skin damagethat has received the first topical formulation. In some embodiments,the activating includes degranulating platelets in the platelet richplasma of the first topical formulation and releasing growth factorsfrom the platelets into the skin of the subject.

In some embodiments, the second formulation is a gel, liquid, cream orlotion. In some embodiments, the second formulation is a liquid whereinthe liquid is applied as a spray such as an aerosol spray or pump spray.In some embodiments, wherein the second formulation is provided as aspray, the second formulation includes Aqua (Water), a plateletactivator, Polysorbate 80, Propylene Glycol, Sodium Chloride, Anthemisnobilis (Chamomile) Flower Oil, Rosa canina (Rose Hip) Fruit Oil, Thymusvulgaris (Thyme) Flower/Leaf Oil, Althaea officinalis (Marshmallow) RootExtract, Hamamelis virginiana (Witch Hazel) Water, Alcohol, Glycerin,Ethylhexylglycerin, Citric Acid, Potassium Sorbate, Phenoxyethanol, orcombinations thereof. In some embodiments, the platelet activator iscalcium chloride.

In some embodiments, the second formulation includes a platelet richplasma activator.

In some embodiments, the platelet rich plasma activator is selected fromthe group consisting of calcium, magnesium, sodium, components of snakevenom, thrombin, collagen, or combinations thereof. The calcium may beprovided as calcium chloride or calcium carbonate, for example. In someembodiments, the second formulation further includes Batroxobin,epinephrine and/or thrombin. In some embodiments, the second formulationincludes 10% calcium (e.g. calcium ion, calcium chloride calciumcarbonate, calcium gluconate). In some embodiments, the secondformulation includes 15%-20% ethanol, 1:4 ratio autologous thrombin toPRP, 10% calcium chloride w/ 10,000 units of bovine thrombin,batroxobin, chitosan, or any combination thereof.

In a third aspect, a kit for treating, preventing, or delaying a skindisorder, a nail disorder, hair loss, or signs of aging is provided. Thekit includes, for example, a first formulation having a topical cosmeticbase, gel, serum, and cream or combination thereof, wherein the firstformulation is admixed with platelet rich plasma, and wherein the firstformulation further includes nutrients, botanicals, preservatives orskin penetrators or a combination thereof; and a spray bottle or aerosolcontainer holding a second formulation, the second formulation includinga platelet rich plasma activator in liquid suspension. In someembodiments, the second formulation includes calcium, magnesium, sodium,components of snake venom or combinations thereof. In some embodiments,the component of snake venom includes Batroxobin. In some embodiments,the second formulation includes 10% calcium (e.g. calcium ion, calciumchloride calcium carbonate, calcium gluconate). In some embodiments, thesecond formulation includes 15%-20% ethanol, 1:4 ratio autologousthrombin to PRP, 10% calcium chloride w/ 10,000 units of bovinethrombin, batroxobin, chitosan, or any combination thereof. In someembodiments, the second formulation includes calcium, magnesium, sodium,components of snake venom or combinations thereof. The calcium may beprovided as calcium chloride or calcium carbonate, for example. In someembodiments, the second formulation further includes Batroxobin,epinephrine and/or thrombin. In some embodiments, the second formulationincludes 10% calcium (e.g. calcium ion, calcium chloride calciumcarbonate, calcium gluconate). In some embodiments, the secondformulation includes 15%-20% ethanol, 1:4 ratio autologous thrombin toPRP, 10% calcium chloride w/ 10,000 units of bovine thrombin,batroxobin, chitosan, or any combination thereof.

In any of the embodiments provided herein, the base composition in thefirst formulation includes aqua (water), Lonicera japonica (honeysuckle)flower extract, glycerin, niacinamide, polyacrylate crosspolymer-6,Lonicera caprifolium (honeysuckle) flower extract, Persea gratissima(avocado) oil, Aloe barbadensis leaf juice, sodium gluconate, sodiumhyaluronate (L), Althaea officinalis (marshmallow) root extract,O-cymen-5-OL, sodium hydroxide, glucose (D), sodium chloride, sodiumcitrate, sodium acetate, sodium bicarbonate, tocopheryl acetate(D-alpha), potassium phosphate, potassium chloride, magnesium chloride,phenoxyethanol, Anthemis nobilis (chamomile) flower oil, Rosmarinusofficinalis (rosemary) leaf oil, Thymus vulgaris (thyme) flower/leafoil, benzyl alcohol, caprylyl glycol, caprylhydroxamic acid, orcombinations thereof, and any of the aforementioned components arepresent in an amount from about 0.001 wt % to about 99% wt %, including0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%, or an amountwithin a range defined by any two of the aforementioned values.

In any of the embodiments of the methods, systems, or compositions, thetopical formulations or base compositions are formulated for cosmeticusage. In some embodiments, the cosmetic usage includes topicalapplication to skin, for treatment, prevention, or delay of a skindisorder, nail disorder, hair loss, or other skin or hair disorder.

In any of the embodiments provided herein, the compositions are providedto a subject together with, or in conjunction with, a skin obstructiondevice, such as electroporation, radiofrequency, ultrasound, highintensity focused ultrasound (HIFU), intense pulsed light (IPL),ablative laser, non-ablative laser, microdermabrasion,hydradermabrasion, iontophoresis, chemical peel, plasma, high velocityair, high velocity aqueous solution, or needling, or a combinationthereof. In some embodiments, the first formulation is applied to asubject, followed by skin obstruction of the skin. In some embodiments,application of a composition followed by skin obstruction improvesupdate of the composition.

Some embodiments provided herein relate to a base composition forprolonging, preserving, or maintaining a biologic. In some embodiments,the composition includes a cell culture reagent, a buffering agent, aviscosity modifying agent, a thymol, and an anti-microbial. In someembodiments, the composition further includes a humectant and magnesiumchloride. In some embodiments, the biological agent is sodiumbicarbonate. In some embodiments, the buffering agent preserves thebiologic at about or above physiological pH. In some embodiments, thethymol is a thymol isomer, a cresol or O-cymen-5-ol. In someembodiments, the anti-microbial is one or more of Lonicera caprifolium(honeysuckle) flower extract and Lonicera japonica (honeysuckle) flowerextract. In some embodiments, the viscosity modifying agent is potassiumchloride, sodium chloride, or both. In some embodiments, the cellculture reagent is fetal bovine serum (FBS) or a growth medium. In someembodiments, the humectant is glucose. In some embodiments, thecomposition includes water, glycerin, niacinamide, polyacrylatecrosspolymer-6, Lonicera caprifolium (honeysuckle) flower extract,Lonicera japonica (honeysuckle) flower extract Persea gratissima(avocado) oil, Aloe barbadensis leaf juice, citric acid, sodiumgluconate, sodium hyaluronate (L), Althaea officinalis (marshmallow)root extract, O-cymen-5-OL, sodium hydroxide, glucose (D), sodiumchloride, sodium citrate, sodium acetate, sodium bicarbonate, tocopherylacetate (D-alpha), potassium phosphate, potassium chloride, magnesiumchloride, phenoxyethanol, Anthemis nobilis (chamomile) flower oil,Rosmarinus officinalis (rosemary) leaf oil, Thymus vulgaris (thyme)flower/leaf oil, benzyl alcohol, caprylyl glycol, caprylhydroxamic acid,or combinations thereof. In some embodiments, the composition includeswater present in an amount of about 92.72 wt %, polyacrylatecrosspolymer-6 present in an amount of about 1.3 wt %, Lonicera japonica(honeysuckle) flower extract present in an amount of about 1.2 wt %,glycerin present in an amount of about 1.2 wt %, niacinamide present inan amount of about 1 wt %, Lonicera caprifolium (honeysuckle) flowerextract present in an amount of about 0.5%, Persea gratissima (avocado)oil present in an amount of about 0.5 wt %, Aloe barbadensis leaf juicepresent in an amount of about 0.5 wt %, citric acid present in an amountof about 0.3 wt %, sodium gluconate present in an amount of about 0.2 wt%, sodium hyaluronate (L) present in an amount of about 0.2 wt %,Althaea officinalis (marshmallow) root extract present in an amount ofabout 0.1 wt %, O-cymen-5-OL present in an amount of about 0.1 wt %,glucose (D) present in an amount of about 0.1 wt %, sodium chloridepresent in an amount of about 0.01 wt %, sodium citrate present in anamount of about 0.01 wt %, sodium acetate present in an amount of about0.01 wt %, sodium bicarbonate present in an amount of about 0.01 wt %,tocopheryl acetate (D-alpha) present in an amount of about 0.01 wt %,potassium phosphate present in an amount of about 0.01 wt %, potassiumchloride present in an amount of about 0.01 wt %, and/or magnesiumchloride present in an amount of about 0.01 wt %. In some embodiments,the composition further includes a biologic. In some embodiments, thebiologic is platelet rich plasma (PRP), fibroblast cells, adiposetissue, adipose derived stromal vascular function (SVF), nanofat,lipoaspirate components, bone marrow derived mesenchymal stem cells,adipose derived stem cells, platelet derived exosomes, adipose derivedexosomes, alpha 2 macroglobulin (A2M), human platelet lysate, orisolated microparticles thereof. In some embodiments, the biologic is alyophilized powder. In some embodiments, the base composition prolongsor enhances stability of the biologic and prolongs or enhances stabilityof components of the biologic. In some embodiments, the base compositioninhibits or prevents activation of the biologic, or wherein the basecomposition maintains, prolongs, preserves, or sustains activity of thebiologic r of already active components of the biologic. In someembodiments, the base composition is formulated for cosmetic usage,wherein the cosmetic usage includes topical application to skin.

Some embodiment provided herein relate to a topical formulation thatincludes a base composition as provided herein, including a cell culturereagent, a buffering agent, a viscosity modifying agent, a thymol, andan anti-microbial and a biologic. In some embodiments, the biologic isplatelet rich plasma (PRP), fibroblast cells, adipose tissue, adiposederived stromal vascular function (SVF), nanofat, lipoaspiratecomponents, bone marrow derived mesenchymal stem cells, adipose derivedstem cells, platelet derived exosomes, adipose derived exosomes, alpha 2macroglobulin (A2M), human platelet lysate, or isolated microparticlesthereof. In some embodiments, the formulation is formulated as a cream,lotion, salve, paste, serum, gel, ointment, liquid, solution, spray,aerosol, or foam. In some embodiments, the topical formulation isformulated for cosmetic usage, wherein the cosmetic usage includestopical application to skin.

Some embodiments provided herein relate to a method of diminishing poresize, diminishing bacteria damage, diminishing red areas, reducing skindamage caused by sun, and maintaining or improving skin tone in asubject in need thereof. In some embodiments, the method includesobtaining whole blood from a subject having an area of skin damage,centrifuging the blood to separate platelet rich plasma from plateletpoor plasma and red blood cells, collecting the platelet rich plasma(PRP), and adding a base composition as disclosed herein to the PRP toform PRP admixed solution, wherein the base composition includes a cellculture reagent, a buffering agent, a viscosity modifying agent, athymol, and an anti-microbial, thereby forming a first topicalformulation, and providing the first topical formulation for applicationto the subject for treatment of the area of skin damage. In someembodiments, the first topical formulation is stored at a temperature ofless than about 10° C., and application of the first topicalformulation, such as application to a subject (including application tothe face of a subject) increases the temperature of the first topicalformulation to at least room temperature. In some embodiments, theincreased temperature of the formulation resulting from application to asubject activates the PRP.

In some embodiments, the method further includes providing a secondtopical formulation for application to the subject over the area of skindamage that has received the first topical formulation, wherein thesecond topical formulation activates the platelet rich plasma. In someembodiments, the base composition maintains activity of the PRP for aperiod of time ranging from 30 to 120 days. In some embodiments, thesubject suffers from skin damage and signs of aging caused by smoking,alcohol, diet, extreme temperatures, chemicals, stress, lack of sleep,poor diet, poor immune system or a combination thereof. In someembodiments, the subject is also suffering from a skin disorder. In someembodiments, the skin disorder causes skin damage and signs of aging. Insome embodiments, the skin disorder is selected from a group consistingof stretch marks (striae), psoriasis, skin cancer, acne, alopecia,carbuncles, dermatitis, eczema, atopic dermatitis, contact dermatitis,seborrheic dermatitis, cradle cap, perioral dermatitis, shingles,ringworm, melisma, impetigo, acne aestivalis (Mallorca acne), acneconglobate, acne cosmetica (cosmetic acne), acne fulminans (acutefebrile ulcerative acne), acne keloidalis nuchae (acne keloidalis,dermatitis papillaris capillitii, folliculitis keloidalis, folliculitiskeloidis nuchae, nuchal keloid acne), adult forehead with scattered redpimples, acne vulgaris, acne mechanica, acne medicamentosa, acnemiliaris necrotica (acne varioliformis), acne vulgaris, acne with facialedema (solid facial edema), blepharophyma, erythrotelangiectatic rosacea(erythematotelangiectatic rosacea, vascular rosacea), excoriated acne(acne excoriee des jeunes filles, Picker's acne), glandular rosacea,gnathophyma, gram-negative rosacea, granulomatous facial dermatitis,rhinophyma, granulomatous perioral dermatitis, halogen acne,hidradenitis suppurativa (acne inversa, pyoderma fistulans significa,Verneuil's disease), idiopathic facial aseptic granuloma, infantileacne, lupoid rosacea (granulomatous rosacea, micropapular tuberculid,rosacea-like tuberculid of Lewandowsky), lupus miliaris disseminatusfaciei, metophyma, neonatal acne (acne infantum, acne neonatorum,neonatal cephalic pustulosis), occupational acne, oil acne, ocularrosacea (ophthalmic rosacea, ophthalmorosacea), otophyma, periorificialdermatitis, persistent edema of rosacea (chronic upper facialerythematous edema, Morbihan's disease, rosaceous lymphedema), phymatousrosacea, pomade acne, papulopustular rosacea (inflammatory rosacea),perifolliculitis capitis abscedens et suffodiens (dissecting cellulitisof the scalp, dissecting folliculitis, perifolliculitis capitisabscedens et suffodiens of Hoffman), perioral dermatitis, periorbitaldermatitis (periocular dermatitis), pyoderma faciale (rosaceafulminans), rhinophyma, rosacea (acne rosacea), rosacea conglobate,synovitis-acne-pustulosis-hyperostosis-osteomyelitis syndrome (SAPHOsyndrome), steroid rosacea, tar acne, skin cancer, tropical acne or acombination thereof. In some embodiments, the subject has wrinkles, acnescars, reduced skin elasticity, sagging skin, increased skin dryness,rashes, redness, translucency, fine lines, loss of radiance, increase inthe dullness of skin, uneven pigmentation, discoloration, blotchiness,scarring, rough and leathery appearance, freckles, moles, actinickeratosis, slower wound healing, easy bruising and tearing, ruddiness,uneven texture, fine lines, age spots, or a combination thereof. In someembodiments, the subject is suffering from hair loss or a hair disorder.In some embodiments, the activating step further includes degranulatingplatelets in the platelet rich plasma of the first topical formulationand releasing growth factors from the platelets into and through theskin of the subject. In some embodiments, the method further includesperforming skin obstruction of the skin over the area of skin damage. Insome embodiments, the skin obstruction includes electroporation,radiofrequency, ultrasound, high intensity focused ultrasound, needling,intense pulsed light (IPL), ablative laser, non-ablative laser,microdermabrasion, hydradermabrasion, iontophoresis, chemical peel,plasma, high velocity air, high velocity aqueous solution, orcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

In order to describe the manner in which the above-recited and otheradvantages and features of the invention can be obtained, a moreparticular description will be rendered by reference to specificembodiments thereof which are illustrated in the appended drawings.Understanding that these drawings depict only typical embodiments andare not therefore to be considered to be limiting of its scope, theinvention will be described and explained with additional specificityand detail through the use of the accompanying drawings in which:

FIGS. 1A-1B depict qualitative improvement of rete peg in skin treatedwith compositions, some of which include platelet rich plasma (PRP).FIG. 1A depicts micrographs showing histopathology for the presence ofrete pegs in control (top) compared to treatment (bottom). FIG. 1Bgraphically shows a quantitative measure of rete peg presence in thecontrol and treatment groups as a measure of stratum basale/stratumganulosum ratio.

FIGS. 2A-2B illustrate micrographs showing collagen type I expression incontrol (FIG. 2A) and treatment (FIG. 2B) groups.

FIG. 3 illustrates intact platelet derived growth factor (PDGF) incompositions at 4° C. assessed over a period of 90 days afterpreparation. PRP control refers to PRP alone, whereas SoME+PRP refers tothe PRP in base composition. Samples were stored at 4° C. and brought toroom temperature at the time of the experiment.

FIG. 4 illustrates intact PDGF in compositions stored at roomtemperature over a period of 90 days. SoME refers to the basecomposition.

FIGS. 5A-5D graphically depict percentage change in assessmentparameters. Various skin assessment parameters are shown for the basecomposition (SoME) compared to PRP+base composition at 4 weeks (FIG. 5A)and 8 weeks (FIG. 5B). The parameters are directly compared at 4 vs 8weeks for PRP+base composition (FIG. 5C) and for the base compositionalone (FIG. 5D).

FIG. 6 graphically illustrates the overall assessment parameters as afunction of percentage change for the base composition alone at 4 and 8weeks and the PRP+base composition at 4 and 8 weeks.

FIGS. 7A-7D graphically depict efficacy parameter scales for the changein skin assessment parameters. The efficacy parameters for weeks 0, 4,and 8 are shown for the base composition alone (FIG. 7A) and for thePRP+base composition (FIG. 7B). The efficacy parameters for 0 and 8weeks are shown for the PRP+base composition (FIG. 7C) and for the basecomposition alone (FIG. 7D).

FIGS. 8-15 show photographs of facial assessment prior to (week 0, leftimage) and after treatment (week 8, right image) in an exampleembodiment provided herein.

DETAILED DESCRIPTION

In the description that follows, the terms should be given their plainand ordinary meaning when read in light of the specification.

“About” as used herein when referring to a measurable value is meant toencompass variations of 20% or 10%, more preferably +5%, even morepreferably +1%, and still more preferably +0.1% from the specifiedvalue.

“Human platelet lysate” (HPL) has its plain and ordinary meaning whenread in light of the specification, and includes, for example, asubstitute supplement for fetal bovine serum in a cell culture. In someembodiments described herein, cells used for treatment are grown inculture that is supplemented with HPL.

HPL can be manufactured by a variety of means that are known to thoseskilled in the art. HPL can be created from single or pooleddonor-donated platelets isolated from whole blood or by apheresis,distributed in a standard platelet collection bag. There are somedifferences between HPL manufacturing protocols, but all can share thesame step of being frozen at very low temperatures and thawed. Thisprocess can be repeated two or three times to cause complete plateletlysis. The resultant HPL can then undergo different manufacturing stepsto achieve multiple grades of HPL.

A common form of HPL undergoes few processing steps, producing a productmade of the supernatant following the freeze/thaw process. The includedclotting factors required to add heparin to the cell culture media toprevent coagulation during incubation.

Another form of HPL is one that can be used in cell culture without theneed of heparin, or any anticoagulant, addition. This grade of HPL goesthrough further manufacturing steps to inhibit the effect the clottingfactors have.

Many labs around the world are creating small amounts of HPL to suittheir laboratory needs. Large-scale manufacturing by pooling manyplatelet donors can be used to mitigate the donor-to-donor variability.Consistency is a top priority for experimental designs to providereproducible results.

Human platelet lysate is commonly used for supplementation of basalmedia in mesenchymal stem cells culture. Prior the use, the pathogeninactivation process is recommended to prevent pathogen transmission.

Furthermore, commercially available human platelet lysate can be used,for example, such as HPL prepared commercially by Mill Creek LifeSciences, Compass Biomedical, Inc., Cook Regentec, Macopharama SA,iBiologics, PL bioscience GmbH and Trinova Biochem GmbH. Product linescan include but are not limited to PLTMax, PLUS™ Stemulate, HumanPlatelet Lysate, XcytePlus, PL_(SOLUTION), PL_(MATRIX) and CRUX RUFAMedia supplements.

PLUS™ human platelet lysate which is commercially available has beenused in some embodiments described herein. The PLUS™ human plateletlysate is a growth factor rich product manufactured by lysing humanplatelets. The base composition can be used as cell culture supplementand the lyophilized product has applications in wound healing.

The product, PLUS™ human platelet lysate, is started from raw materialwhich is expired transfusion platelets sourced from FDA-registered andISO-registered blood banks. The raw material inventories, which are theplatelets from the blood banks, are then selected for the productioninitiation, in which the units are identified for thawing. The thawedpool of platelets are then pooled and transferred into a large transferbag for serum conversion. The material is then further centrifuged andpooled in a large container for distribution into bottles or cry bags.The raw material is then tested for Human Immunodeficiency Virus I/II,Human T-Lymphotropic Virus, Hepatitis B Virus, Hepatitis C Virus,Syphilis, West Nile Virus and Trypanosoma cruzi. Testing is carried outin a CLIA certified test laboratory. After testing, the raw material isthen manufactured through a GMP closed-loop production system for makingof the final product, which is a liquid supplement for cell culture,lyophilized product for wound healing applications. The final product isthen tested for bacterial & fungal contamination, endotoxin, mycoplasma,MSC expansion, pH, osmolality, hemoglobin, total protein and bloodchemistry. The material is filter sterilized. If the material is to bemade into a lyophilized powder, the base composition is then sterilelyophilized. The full GMP manufacturing system is in place with incomingproduct inspection, employee training, SOPs, record keeping and qualitycontrol.

The final product as a base composition is available as a researchproduct for cell culture supplementation, in which it is provided incryo-bags and bottles. A certificate of Analysis is always included withall products.

The final product as a lyophilized powder has minimal change in activityupon lyophilization/reconstitution and is stable at room temperature.The powder can be reconstituted in various solutions and gels (water,PPP, alginate). Currently, the powder is being testing for advancedwound healing applications.

The final product of PLUS™ human platelet lysate has several featuresand benefits for its use. For example, the human derived, growth factorrich, serum or powder supplement can support in vitro propagation ofMSCs, keratinocytes, fibroblasts and other cell types. As such, thePLUS™ human platelet lysate has all the benefits of PRP without theinconvenience of needles and blood draws. Furthermore, the plateletshave been sourced from FDA-registered blood banks, so that rigorousserology testing and infectious disease screenings have already beenperformed. Additionally the large-scale GMP manufacturing has alreadybeen established. There are over 100 donor platelet units pooled foreach lot, there is consistent product with minimal lot-to-lot variation,and each lot has been tested for MSC expansion, total protein content,pH and several other parameters.

The PLUS™ human platelet lysate is considered very safe to use. Thematerial sourced for the product has come from donors who have beenstringently screened and tested for infectious diseases. The plateletsare then collected and frozen immediately after a five day expiration.Other safety features include manufacturing the product within a fullyclosed loop system to prevent exposure to sources of potentialcontamination and then there is an additional rigorous testing of thefinal product to ensure that there are no bacterial and fungalcontaminations, endotoxins and mycoplasma.

PLUS™ human platelet lysate has several deliverable options. It can bedelivered as a cell culture supplement (GMP Plus™), as Plus™ lyophilizedin individual packages for reconstitution with platelet poor plasma(PPP), as Plus™ lyophilized and shipped in bulk volumes of 500 mL to 1L, or as a kit with lyophilized GMP Plus™ in 10 mL sterile glass vialwith a crimp seal, vial adapter and a syringe. The prices for thesupplements depending on volume and product can range from $275 to $845(for example, 250 mL of GMP Plus™ Cell Culture Supplement). For thelyophilized packages, the kits can come with the instructions for thereconstitution into a liquid formula. The typical lot sizes for GMPPlus™ are 20 liters although custom lots can be made for up to 50liters. For consistency in lab tests, lot reservations can be made anddelivery for orders for up to 10 liters are immediate.

In some embodiments, a system, method, composition, or kit is providedfor preparation of a first topical formulation. In some embodiments, auser obtains a sample of whole blood from the user himself, such that abiologic that is subsequently obtained is autologous. In someembodiments, the user processes the whole blood to obtain a biologic. Insome embodiments, the biologic is purified and submitted tolyophilization to obtain a lyophilized biologic powder. Lyophilizationtechniques can include, for example, freeze drying, vacuumlyophilization, or other lyophilization techniques. In some embodiments,the lyophilized biologic powder is weighed and aliquoted in specificamounts in separate vessels, and stored for later usage. In someembodiments, the lyophilized biologic is dissolved by adding to thevessel a dissolution solution, which may include any aqueous solutioncapable of dissolving or solubilizing the lyophilized powder. Forexample, a dissolution solution may include water, sodium chloride,phosphate buffered saline, saline, a buffer, a salt solution, or anyother solubilizing agent, thereby generating a biologic dissolved insolution. In some embodiments, the biologic solution is then added to abase composition as described herein, such that the base compositionpreserves, extends, prolongs, maintains, or sustains the biologic. Insome embodiments, the base composition preserves, extends, prolongs,maintains, or sustains components of biologics, such as growth factors,proteins, nucleic acids, or other biomolecules associated with thebiologic. In some embodiments, the base composition preserves thebiologic in the solution for a period of 30, 45, 60, 75, 90, 105, or 120days, or longer, such that the biologic formulation exhibitstherapeutically effective results when applied on the skin of thesubject over a treatment period. In some embodiments, upon completeusage of the biologic formulation, or after a period of time wherein thebiologic or the components thereof are no longer viable, a secondaliquot of lyophilized biologic powder may be dissolved and combinedwith the base composition. In some embodiments, the lyophilized PRPpowder remains stable for a period of longer than 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30months.

The human platelet lysate can be formed from but not limited to plateletrich plasma (PRP), pooled platelets from humans and culturedmegakaryocytes from stem cell expansion technology. In some embodimentsdescribed herein, HPL is from a commercial source. In some embodimentsdescribed herein, the human platelet lysate is prepared in thelaboratory from platelet rich plasma (PRP), pooled platelets from humansor cultured megakaryocytes from stem cell expansion technology.

In some embodiments herein, the platelet rich plasma and the humanplatelet lysate can be drawn from the subject that is in need oftreatment, prevention, or delay of a skin disorder, nail disorder, orhair disorder and then used to treat the subject themselves. Thus, thesubject can be the source of their own products and their owntreatments.

Platelets are composed of a cytoskeleton and intracellular structuressuch as glycogen, lysosomes, and two granules, the dense granule and thealpha-granule. The dense granule comprises adenosine diphosphate (ADP),adenosine triphosphate (ATP), serotonin, and calcium, while thealpha-granule comprises clotting factors, growth factors, and proteins.Embodiments of the present disclosure include compositions capable ofmaintaining the enclosed cellular components active for extended timeperiods and obtaining platelets in sufficient quantity to be consideredplatelet rich plasma.

The normal platelet count in human blood is 150,000-350,000/microliter.“Platelet rich plasma” (PRP) has its plain and ordinary meaning whenread in light of the specification, and includes, for example, a bloodplasma that has been enriched with platelets. PRP is a component ofblood (plasma) with concentrations of platelets above normal values.Thus, PRP refers to a sample having a platelet count of greater than350,000 platelets per microliter, such as 350,000, 400,000, 450,000,500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000,900,000, 950,000 or greater than 1,000,000 platelets per microliter, orin an amount within a range defined by any two of the aforementionedvalues. PRP may further include an amount of platelets greater than anormal platelet count with a full complement of clotting factors. Sevenkey growth factors may be present in PRP, including, for example,platelet derived growth factors (PDGFaa, PDGFbb, PDGFab), transforminggrowth factors (TGF-b1, TGFb2), vascular endothelial growth factor(VEGF), and epithelial growth factor (EGF). These growth factors arefound within a normal clot composed of fibrin, fibronectin, andvitronectin, which are cell adhesion molecules required for cellmigration, as seen in wound healing. Cellular mitogenesis andangiogenesis are both upregulated by PRP, making it useful in facialrejuvenation.

After injury, platelets are on the front line of the healing responseand play a critical role by releasing growth factors. These growthfactors influence tissue repair in a variety of different cell typesincluding tendon, muscle and cartilage cells. PRP was first used indental and oral surgery to improve soft tissue healing in the 1990s. Itsusage in the treatment of musculoskeletal injuries and sports medicinehas increased over the past decade. PRP has been used as an injectablematerial for enhanced healing, hair growth, or for facial rejuvenation.

In some embodiments, autologous PRP is obtained from freshly drawn bloodfrom a patient with an added anticoagulant and the sample then undergoesa series of two centrifugation (spin) steps. The first spin, known asthe hard spin, separates the red blood cells from the plasma containingthe platelets, white blood cells, and clotting factors. Three layersresult from the hard spin: an upper layer containing platelets and whiteblood cells, a middle layer known as the buffy coat containing whiteblood cells, and a bottom layer containing red blood cells. The redblood cell layer is removed and discarded. The second spin, known as thesoft spin, separates the platelet rich plasma in the bottom of the tubefrom the platelet poor plasma (PPP) in the top of the tube by removingmore red blood cells. Proper preparation and centrifuge technique iscritical to obtaining high quality active PRP. The literature haspointed to some preparations of PRP having a lack of biological effectwhich may be due to poor PRP processing or inadequate standardlaboratory centrifuges that cannot properly prepare PRP rather than thespecialized FDA cleared equipment with validated processes.

As a concentrated source of autologous platelets, PRP contains andreleases through several different growth factors and other cytokinesthat stimulate healing of bone and soft tissue. The components of PRPcan include but is not limited to platelet-derived growth factor,transforming growth factor beta, fibroblast growth factor, insulin-likegrowth factor 1, insulin-like growth factor 2, vascular endothelialgrowth factor, epidermal growth factor, Interleukin 8, keratinocytegrowth factor, and connective tissue growth factor.

PRP can be prepared by collection of the patient's whole blood (that isanticoagulated with citrate dextrose) before undergoing two stages ofcentrifugation designed to separate the PRP aliquot from platelet-poorplasma and red blood cells. The PRP is denser than the platelet poorplasma and is then collected. In humans, the typical baseline bloodplatelet count is approximately 200,000 per μL; therapeutic PRPconcentrates the platelets by roughly five-fold. The PRP can then beused to prepare human platelet lysate. Typically 20 mL of whole bloodmay be drawn to generate at least 3 mL of PRP. Typically 60 mL of wholeblood may be drawn to generate at least 7 to 10 mL of PRP. In someembodiments, a blood draw for preparing PRP may be in an amount rangingfrom 1 mL to 60 mL, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, or 60 mL of blood, or in an amount within arange defined by any two of the aforementioned values. As PRP in theembodiments here are originating from the same subject that is treatedby the method embodiments herein, the safety concerns are minimal. Insome embodiments herein, the platelet rich plasma are from a subjectthat is being treated for a skin disorder, nail disorder, hair disorder,or signs of aging. In some embodiments, the whole blood is obtained froma subject having skin damage and/or signs of aging, the whole blood iscentrifuged to separate platelet rich plasma from platelet poor plasmaand red blood cells and the platelet rich plasma (PRP) is collectedafter centrifugation. The centrifuged blood also has a layer of buffycoat, which is the fraction of anticoagulated blood that contains mostof the white blood cells and some platelets. In some embodiments, theplatelet rich plasma is used to manufacture platelet lysate. In someembodiments, platelet lysate may be produced by undergoing a freeze andthaw process of the PRP.

Because PRP is autologous, concerns about immune rejection are anon-issue. Growth factors can function by activating a cytoplasmicsignal that promotes normal gene expression. PRP can include the samematerials present in the blood stream that induce clotting, except inhigher concentration. In some embodiments, PRP works throughdegranulation of the alpha granules in platelets, which contain thegrowth factors. PRP can be collected in an anticoagulated state for thegrowth factors to remain active explaining the need to draw the bloodinto a tube containing sodium citrate. The biologically active cellularcomponents such as the growth factors can be preserved within theplatelet when the platelet is held in an inactive state; prematuredegranulation does not occur and therefore the cellular components areheld within the protective enclosure of the platelet. Upon activation ofthe platelet, the granules can fuse to the cell membrane, thedegranulation process, activating the secretory growth factors, whichcan bind to the transmembrane receptors of target cells, such asmesenchymal stem cells, fibroblasts, endothelial cells, and epidermalcells. This binding can activate intracellular signal proteins thatexpress a gene sequence directing cellular proliferation, collagensynthesis, extracellular matrix formation, and numerous other pathwaysto promote healing and repair processes. Damaged platelets with degradedor non-viable cellular components can be incapable of inducing thisresponse.

Aging of the skin can be classified into two components: intrinsic andextrinsic aging. As the names imply, intrinsic aging is due togenetically controlled senescence and extrinsic aging is due toenvironmental factors superimposed on intrinsic aging. Platelet richplasma (PRP) is obtained from plasma by centrifuging the blood toconcentrate the stem cell materials. The PRP can be injected intovarious skin areas to improve appearance.

“Skin damage” has its plain and ordinary meaning when read in light ofthe specification, and includes, for example, damage to the skin thatcan be caused by aging, sun damage, cancer, skin disorder or skindiseases that can cause irritation of the skin. “Without being limiting,the “skin diseases” and/or “skin disorders” can include acne aestivalis(Mallorca acne), acne conglobate, acne cosmetica (cosmetic acne), acnefulminans (acute febrile ulcerative acne), acne keloidalis nuchae (acnekeloidalis, dermatitis papillaris capillitii, folliculitis keloidalis,folliculitis keloidis nuchae, nuchal keloid acne), adult forehead withscattered red pimples, acne vulgaris, acne mechanica, acnemedicamentosa, acne miliaris necrotica (acne varioliformis), acnevulgaris, acne with facial edema (solid facial edema), blepharophyma,erythrotelangiectatic rosacea (erythematotelangiectatic rosacea,vascular rosacea), excoriated acne (acne excoriee des jeunes filles,Picker's acne), glandular rosacea, gnathophyma, gram-negative rosacea,granulomatous facial dermatitis, adult male with a large, red, bulbousnose, rhinophyma, granulomatous perioral dermatitis, halogen acne,hidradenitis suppurativa (acne inversa, pyoderma fistulans significa,Verneuil's disease), idiopathic facial aseptic granuloma, infantileacne, lupoid rosacea (granulomatous rosacea, micropapular tuberculid,rosacea-like tuberculid of Lewandowsky), lupus miliaris disseminatusfaciei, metophyma, neonatal acne (acne infantum, acne neonatorum,neonatal cephalic pustulosis), occupational acne, oil acne, ocularrosacea (ophthalmic rosacea, ophthalmorosacea), otophyma, periorificialdermatitis, persistent edema of rosacea (chronic upper facialerythematous edema, Morbihan's disease, rosaceous lymphedema), phymatousrosacea, pomade acne, papulopustular rosacea (inflammatory rosacea),perifolliculitis capitis abscedens et suffodiens (dissecting cellulitisof the scalp, dissecting folliculitis, perifolliculitis capitisabscedens et suffodiens of Hoffman), perioral dermatitis, periorbitaldermatitis (periocular dermatitis), pyoderma faciale (rosaceafulminans), rhinophyma, rosacea (acne rosacea), rosacea conglobate,synovitis-acne-pustulosis-hyperostosis-osteomyelitis syndrome (SAPHOsyndrome), steroid rosacea, tar acne, akin cancer and tropical acne. Insome embodiments described herein, a method of treating a subject inneed is provided. The subject can have acne aestivalis (Mallorca acne),acne conglobate, acne cosmetica (cosmetic acne), acne fulminans (acutefebrile ulcerative acne), acne keloidalis nuchae (acne keloidalis,dermatitis papillaris capillitii, folliculitis keloidalis, folliculitiskeloidis nuchae, nuchal keloid acne), adult forehead with scattered redpimples, acne vulgaris, acne mechanica, acne medicamentosa, acnemiliaris necrotica (acne varioliformis), acne vulgaris, acne with facialedema (solid facial edema), blepharophyma, erythrotelangiectatic rosacea(erythematotelangiectatic rosacea, vascular rosacea), excoriated acne(acne excoriee des jeunes filles, Picker's acne), glandular rosacea,gnathophyma, gram-negative rosacea, granulomatous facial dermatitis,adult male with a large, red, bulbous nose, rhinophyma, granulomatousperioral dermatitis, halogen acne, hidradenitis suppurativa (acneinversa, pyoderma fistulans significa, Verneuil's disease), idiopathicfacial aseptic granuloma, infantile acne, lupoid rosacea (granulomatousrosacea, micropapular tuberculid, rosacea-like tuberculid ofLewandowsky), lupus miliaris disseminatus faciei, metophyma, neonatalacne (acne infantum, acne neonatorum, neonatal cephalic pustulosis),occupational acne, oil acne, ocular rosacea (ophthalmic rosacea,ophthalmorosacea), otophyma, periorificial dermatitis, persistent edemaof rosacea (chronic upper facial erythematous edema, Morbihan's disease,rosaceous lymphedema), phymatous rosacea, pomade acne, papulopustularrosacea (inflammatory rosacea), perifolliculitis capitis abscedens etsuffodiens (dissecting cellulitis of the scalp, dissecting folliculitis,perifolliculitis capitis abscedens et suffodiens of Hoffman), perioraldermatitis, periorbital dermatitis (periocular dermatitis), pyodermafaciale (rosacea fulminans), rhinophyma, rosacea (acne rosacea), rosaceaconglobate, synovitis-acne-pustulosis-hyperostosis-osteomyelitissyndrome (SAPHO syndrome), steroid rosacea, tar acne, akin cancer and/ortropical acne.

In some embodiments, skin damage is a result of surgery or lasertreatment. Thus, in some embodiments, the formulations provided hereinare used post-operatively or post-laser treatment to improve compromisedskin. Without wishing to be bound by theory, treatment of the skin asdescribed herein may follow a pathway of allowing subcellular componentsor microparticles to penetrate skin and release microRNAs for improvingdamaged skin cells. In some embodiments, the PRP formulation in the basecomposition is applied topically to a subject for treatment of postlaser or post-operative procedures to enhancing healing effects, forminimizing scar tissue formation, or for reducing the appearance ofscars.

“Hair and scalp disorders” have their plain and ordinary meaning whenread in light of the specification, and includes, for example, diseasesthat affect the hair and skin on the scalp and are also describedherein. Diseases that affect hair and scalp can include but are notlimited to alopecia, androgenic alopecia, hirsutism, hair shaftdisorders, inflammation, acromegaly, eczema, stretch marks (striae),psoriasis, impetigo, atopic dermatitis, darier disease, hair loss, andfolliculitis. Top causes for scalp disorders can include but is notlimited to acromegaly, atopic dermatitis, darier disease, eczema,fragile X syndrome, impetigo, pachydermoperiostosis, psoriasis andRosenthal-Kloepfer syndrome. In some embodiments described herein, amethod of treating a subject in need is provided. The subject can have adisease affecting the skin and scalp. In some embodiments the subjectsuffers from alopecia, androgenic alopecia, hirsutism, hair shaftdisorders, inflammation, acromegaly, eczema, stretch marks (striae),psoriasis, impetigo, atopic dermatitis, darier disease, and/orfolliculitis. In some embodiments, the subject suffers from acromegaly,atopic dermatitis, darier disease, eczema, fragile X syndrome, impetigo,pachydermoperiostosis, psoriasis and/or Rosenthal-Kloepfer syndrome. Insome embodiments, the treating includes administering a formulation tothe subject in need. In some embodiments, the formulation is within ahair cream, a hair gel, a scalp lotion, a shampoo, conditioner, hairspray or a hair mousse.

“Nail diseases” have their plain and ordinary meaning when read in lightof the specification, and includes, for example, disorders or diseasesthat affect the nail, nail bed, cuticle region and the surrounding skinand are also described herein. Diseases that affect the nail andsurrounding skin area such as the cuticle can lead to infection orinflammation that could require medical assistance. Diseases that infectthe nail, nail bed and/or cuticle can include but is not limited toonychia, onychocryptosis, onychodystophy, onychogryposis, onycholysis,onychomadesis, onychomycosis, tinea unguium, onychophosis, onychoptosis,onchorrhexis, paronychia, Koilonychia, subungual hematoma,onychomatricoma, nail pemphigus, erythronychia and melanonychia. In someembodiments, the methods provided herein improve fingernail texture. Insome embodiments, the methods provided herein improve a nail disorder, anail disease, or a nail infection. In some embodiments described herein,a method of treating a subject in need is provided. The subject can havea disease affecting the nails, nail bed and/or cuticles. In someembodiments the subject suffers from alopecia, androgenic alopecia,hirsutism, hair shaft disorders, inflammation, acromegaly, eczema,stretch marks (striae), psoriasis, impetigo, atopic dermatitis, darierdisease, and/or folliculitis. In some embodiments, the subject suffersfrom onychia, onchyocryptosis, onychodystophy, onychogryposis,onycholysis, onychomadesis, onychomycosis, tinea unguium, onychophosis,onychoptosis, onchorrhexis, paronychia, Koilonychia, subungual hematoma,onychomatricoma, nail pemphigus, erythronychia and/or melanonychia. Insome embodiments, the treating includes administering a formulation tothe subject in need. In some embodiments, the formulation is within askin cream, a lotion or a cuticle cream.

“Inflammation” has its plain and ordinary meaning when read in light ofthe specification, and includes, for example, a biological response of abody tissue to harmful stimuli. The harmful stimuli can include but isnot limited to pathogens, bacteria, viruses, fungi, damaged cells andother irritants that are known to those skilled in the art. Inflammationcan be a protective immune response that can involve, for example,immune cells, white blood cells, blood vessels, molecular mediators, andother small molecules. Signs of inflammation can include but is notlimited to pain, heat, swelling, and/or loss of function. Inflammationcan be acute or chronic. In some embodiments described herein, aformation is provided for the treatment of inflammation. The formulationcan include cells manufactured by the methods described herein. In someembodiments, the subject suffers from inflammation. In some embodiments,the inflammation is on the skin of the scalp, and nail area such as thecuticles.

“Tear troughs” have their plain and ordinary meaning when read in lightof the specification, and includes, for example, the hollow indentationsthat are beneath the eye. Tear troughs can also appear due to saggyskin. Saggy skin can be caused by aging, loss of collagen and elastinunderneath the skin tissues and sudden weight loss. For cosmeticpurposes saggy skin is usually treated around the neck region and facialregions such as the laugh lines, tear troughs and the jaw jowls.

“Micronize” has its plain and ordinary meaning when read in light of thespecification, and includes, for example, breaking of a substance intovery fine particles, for example, into particles that are only a fewmicrons in diameter. Micronizing can be performed by a micronizer.Examples of several other types of commercial type micronizers foraspirate and organ tissue are known to those skilled in the art. In someembodiments herein, it is contemplated that tissue from the subject maybe micronized in order to provide cells or tissue that can be used inthe embodiments of the formulations herein.

“Pharmaceutical vehicle” has its plain and ordinary meaning when read inlight of the specification, and includes, for example, an inertsubstance with which a medication is mixed to facilitate measurement andadministration of the pharmaceutical formulation. In some embodiments,the pharmaceutical vehicle may allow growth factors from granulation togo through the skin to help heal damaged skin or alleviate the symptomsof a skin disorder.

“Growth media” or “media,” has its plain and ordinary meaning when readin light of the specification, and includes, for example, a solid orliquid designed to support the growth of microorganisms or cells. Commongrowth media for cells, for example, are nutrient broths and agarplates; specialized media are sometimes required for cell culturegrowth. In the embodiments herein, the media is needed for cell culturegrowth and expansion. Without being limiting the growth media can be acommercially obtainable growth media that is specialized for eukaryoticcells or mammalian cells. There are several types of media. Withoutbeing limiting, this can include, nutrient media, minimal media,selective media, differential media, transport media, and enrichedmedia. The media can be obtained commercially or can easily bemanufactured by one with skill in the art. Without being limiting, thecell culture media comprises a carbon source, amino acids, vitamins,minerals, antioxidants, a balanced salt solution, and/or a buffer tomaintain a balanced pH in the media during cell growth. Without beinglimiting, commercially available nutrient media specifically formammalian cells can be made by ThermoFisher Scientific (DMEM, IMDM, RPMI1640, MEM, OPti-MEM medium, DMEM/F-12, GluaMax, Advanced media, Recoveryfreezing medium, F10 Nutrient mixture, Ham's F12 Nutrient mixture, Media199, Opti-MEM, SensiCell Media for Sensitive cells, BME, Stem cell media(Essential 8™, StemPro™, MSC SFM media)), EMD-Millipore (Cellvento™,Cellvento™ BHK cell culture medium, Cellvento™ CHO cell culture mediaplatform, Customized Cell Culture Media, Classical Cell Culture Media),Fisher Scientific (Corning™ Cellgro™ Minimal Essential Medium Eaglewithout Glutamine, Corning™ Cellgro™ DMEM with L-glucose and sodiumpyruvate), Stemcell™ Technologies (mTESR™ 1, MethoCult™ H4034 Optimim,BrainPhys™), Life Technologies, Invitrogen™ (RPMI 1640 medium) and GEHealthcare (AciCHO P). Cell media for mammalian cells can also be madeby those skilled in the art. Without being limiting this can includeusing recipes for Ham's Medium, which contains all the amino acids,purines and pyrimidines for the synthesis of nucleotides, precursors forsynthesizing phospholipids, vitamins, coenzyme lipoic acid, glucose andinorganic ions (sodium, potassium, calcium, copper, zinc and cobalt).The cell media can further be modified with growth factors and proteins.Media can also be optimized for improving cell recovery and activityand/or viability of cells during a freeze-thaw step for the cells. Insome embodiments, the media includes preservatives and nutrients tomaintain the activity and/or viability or platelet cells and/orfibroblast cells. The term mineral has its ordinary meaning asunderstood in light of the specification and refers to any mineral. Nonlimiting examples of minerals include: iodine, manganese, potassium,sodium, selenium, chromium, molybdenum, calcium, phosphorus, zinc, iron,or copper, or any salt form thereof.

“Media Supplements” have their plain and ordinary meaning when read inlight of the specification, and includes, for example, elements or asupplement that are used to help mammalian cells produce proteins, andcan be used to customize the growth conditions of the cells as providedherein, by improving cell activity and/or viability and maintaining thegrowth of the cells. Without being limiting, the supplements can includeamino acids, 2-mercaptoethanol, lipids, MEM vitamin solutions(commercially made or produced in a laboratory), BSA, human keratinocytegrowth supplements, human melanocyte growth supplements, microvasculargrowth supplements, cholesterol supplements, transferrin, sodiumpyruvate and/or vitamins. In some embodiments herein, a method of makinga cell for treatment of a subject is provided, the method comprisingobtaining cells from the subject, placing cells in growth media, whereinthe growth media comprises human platelet lysate and growing the cellsup to confluency. In some embodiments, the growth media comprises mediasupplements.

“Base” has its plain and ordinary meaning when read in light of thespecification, and includes, for example, a base for use in aformulation that comprises cells. A base may also be a formulation inwhich the cells are mixed in. The base may be an emulsion base. In someembodiments, cells or platelet rich plasma is admixed with a base toproduce a first formulation that can be applied to the skin.

“Admix” has its plain and ordinary meaning when read in light of thespecification, and includes, for example, mixing one product with someother product. In the embodiments herein, cells are admixed with acosmetic base or formulation. In some embodiments, platelet rich plasmais admixed with a base, such as a cosmetic base or a formulationcomprising a pharmaceutical vehicle.

“Maintaining youthful appearance” has its plain and ordinary meaningwhen read in light of the specification, and includes, for example,decreasing the signs of aging such as fine lines, wrinkles, saggy skin,dullness, for example. This may also indicate that one can preventfurther damage and signs of aging.

“Signs of aging” has its plain and ordinary meaning when read in lightof the specification, and includes, for example, the appearance of theskin that may occur due to skin damage, exposure, dehydration, aging,illness, chemical treatment, harsh treatment of skin and sun exposure,for example. Signs of aging can include, wrinkles, acne scarring, acnescars, reduced skin elasticity, sagging skin, increased skin dryness,rashes, redness, translucency, fine lines, loss of radiance, increase inthe dullness of skin, uneven pigmentation, discoloration, blotchiness,scarring, rough and leathery appearance, freckles, moles, actinickeratosis, slower wound healing, easy bruising and tearing, ruddiness,uneven texture, fine lines, age spots, or a combination thereof.

“Platelet rich activator” has its plain and ordinary meaning when readin light of the specification, and includes, for example, a formulationthat allows platelets to degranulate. “Degranulate” as described hereinis a process of losing or releasing granules of a substance. Thiscellular process allows cytoplasmic granules within the cell to secretetheir contents to the outside of the cell. In some embodiments, thebiologic contents are then freed from the cell, enabling the biologiccontents to penetrate through the skin of a subject, and thereby used asa skin rejuvenation agent. In some embodiments, the platelet richactivator is administered as a lotion, liquid or a gel. In someembodiments, the platelet rich activator is within a liquid, wherein theliquid is administered by a spray onto skin that has been firstadministered a first formulation comprising adding a topical cosmeticbase, gel, serum, and cream or combination thereof and platelet richplasma. In some embodiments, the first formulation further comprises atopical cosmetic base. In some embodiments, platelet rich activator iswithin a second formulation, and is administered as a liquid spray. Insome embodiments, the platelet rich plasma activator comprises calcium,CaCl₂), Calcium carbonate, thrombin, autologous thrombin, bovinethrombin, collagen type I, magnesium, sodium, components of snake venom,or any other agent capable of activating platelets or allowing plateletsto degranulate, or combinations thereof. Concentrations of platelet richactivators for use are known to those of skill in the art and can befound in Yuan et al. (Osteoarthritis and Cartilage 21 (2013) 1627e1637),Marx et al (Oral Medicine, Vo., 85, no. 6, June 1998, pp. 638-647),Mazzucco et al. (ISBT Science Series (2007) 2, 272-281), Ehrenfest etal. (Trends in Biotechnology Vol. 27 No. 3, pp. 158-167), and Everts etal. (JECT. 2006; 38:174-187) (all references incorporated in theirentireties herein). In some embodiments, the component of snake venomcomprises Batroxobin. In some embodiments, the second formulationcomprises 10% calcium (e.g. calcium ion, calcium chloride calciumcarbonate, calcium gluconate). In some embodiments, the secondformulation comprises 15%-20% ethanol, 1:4 ratio autologous thrombin toPRP, 10% calcium chloride w/ 10,000 units of bovine thrombin,batroxobin, chitosan, or any combination thereof. In some embodiments,wherein the second formulation is provided as a spray, the secondformulation comprises Aqua (Water), a platelet activator, Polysorbate80, Propylene Glycol, Sodium Chloride, Anthemis nobilis (Chamomile)Flower Oil, Rosa canina (Rose Hip) Fruit Oil, Thymus vulgaris (Thyme)Flower/Leaf Oil, Althaea officinalis (Marshmallow) Root Extract,Hamamelis virginiana (Witch Hazel) Water, Alcohol, Glycerin,Ethylhexylglycerin, Citric Acid, Potassium Sorbate, Phenoxyethanol. Insome embodiments, the platelet activator is calcium chloride.

“Collagen” has its plain and ordinary meaning when read in light of thespecification, and includes, for example, the main structural proteinthat may be found in the extracellular space in the various connectivetissues in animal bodies. In some embodiments, the second formulationwhich comprises the platelet rich plasma activator comprises collagen.

“Nanopearls” has its plain and ordinary meaning when read in light ofthe specification, and includes, for example, pharmaceutical lipidnanoparticles to allow nanoemulsions.

“Snake venom” has its plain and ordinary meaning when read in light ofthe specification, and includes, for example, a highly modified saliva,which contains zootoxins that facilitate the immobilization and thedigestion of a snake's prey. The main toxins and proteins in snake venommay have, but is not limited to dehydrogenase lactate, L-amino-acidoxidase, Catalase, Alanine amino transferase, Phospholipase A2,Lysophospholipase, Acetylcholinesterase, Alkaline phosphatase, Acidphosphatase, 5′-Nucleotidase, Phosphodiesterase, Deoxyribonuclease,Ribonuclease 1, Adenosine triphosphatase, Amylase, Hyaluronidase,NAD-Nucleotidase, Kininogenase, Factor-X activator, Heparinase,α-Fibrinogenase, 0-Fibrinogenase, Fibrinolytic enzyme, Prothrombinactivator, Collagenase, Elastase and Glucosamine ammonium lyase. In someembodiments, the snake venom comprises oxydoreductases, transferases,hydrolases, or lyases or a combination thereof. In some embodiments, thesnake venom comprises dehydrogenase lactate, L-amino-acid oxidase,Catalase, Alanine amino transferase, Phospholipase A2,Lysophospholipase, Acetylcholinesterase, Alkaline phosphatase, Acidphosphatase, 5′-Nucleotidase, Phosphodiesterase, Deoxyribonuclease,Ribonuclease 1, Adenosine triphosphatase, Amylase, Hyaluronidase,NAD-Nucleotidase, Kininogenase, Factor-X activator, Heparinase,α-Fibrinogenase, β-Fibrinogenase, Fibrinolytic enzyme, Prothrombinactivator, Collagenase, Elastase or Glucosamine ammonium lyase orcombinations thereof. In some embodiments, the snake venom comprisesα-neurotoxins, β-neurotoxins, κ-Toxins, Dendrotoxins, Cardiotoxins,Myotoxins, Sarafotoxins or Hemorrhagins, or combinations thereof. Insome embodiments the snake venom comprises α-Bungarotoxin, α-toxin,erabutoxin, cobratoxin, Notexin, ammodytoxin, β-Bungarotoxin, crotoxin,taipoxin, κ-Toxin, Dendrotoxin, toxins I and K, y-Toxin, cardiotoxin,cytotoxin, Myotoxin-a, crotamine, Sarafotoxin a, Sarafotoxin b,Sarafotoxin c, Phospholipase A2, mucrotoxin A, hemorrhagic toxins, HT1or HT2 or combinations thereof. In some embodiments, the component ofsnake venom comprises Batroxobin.

In some embodiments, the compositions described herein relate to a basecomposition that preserves, extends the life of, enhances, maintains,sustains, or otherwise prolongs a biologic, such as a cell, a protein,an antibody, blood, serum, plasma, or components thereof, or otherbiological extracts. In some embodiments, the biologic comprisesplatelet rich plasma (PRP), fibroblast cells, adipose tissue, adiposederived stromal vascular function (SVF), nanofat, lipoaspiratecomponents, bone marrow derived mesenchymal stem cells, adipose derivedstem cells, platelet derived exosomes, adipose derived exosomes, alpha 2macroglobulin (A2M), human platelet lysate, or isolated microparticlesthereof, or combinations thereof. In some embodiments, the basecomposition is mixed with or combined with a biologic to form a firstformulation. In some embodiments, the first formulation preserves,extends the life of, enhances, maintains, sustains, or otherwiseprolongs a biologic, for example, for a period of more than 5, 10, 15,20, 30, 40, 50, 60, 70, 80, 90 or 120 days, or an amount of days withina range defined by any two of the aforementioned values. In someembodiments, the second formulation causes degranulation of theplatelets in the first formulation to allow growth factors andbiomolecules from the granules to be released into and through the skinto promote healing of a skin disorder and to prevent or treat signs ofaging.

In some embodiments, the base composition preserves, extends the lifeof, enhances, maintains, sustains, or otherwise prolongs components of abiologic. A component of a biologic may include, for example, growthfactors, proteins, nucleic acids, or other biomolecules that areassociated with, excreted from, or derived from a biologic.

Numerous challenges present for preserving and maintaining platelet richplasma (PRP) for purposes of preparing a product. Among these challengesis the difficulty of platelet preservation, growth factor preservation,and transdermal drug delivery. In product development phases, developersfocus on platelet preservation/growth factor preservation with the useof platelet additive solutions (PAS) to inhibit platelet activation andprolong growth factor degradation. Prior research suggests that the useof platelet specific components may correspond to at least 18 to 20 daysof PRP storage and preservation at 22° C. Furthermore, 21-day oldplatelets are as proliferative in growth factor activity as two-day oldplatelets, which suggests that expired liquid preserved platelets canstill house growth factors in the alpha granule after expiration. Theseteachings suggest that platelets may be viable for 18 to 20 days, andafter expiration may still house the growth factors for at least 21additional days, such that growth factor activity may remain for atleast 39 days. Once the growth factors are in solution, proteindegradation takes another two to three weeks, resulting in a totalproduct shelf life of 53 days. Embodiments provided herein prolong theability to preserve platelet and growth factors, thereby improvingproduct development and longevity, and significantly enhancing productuse.

“Botanicals” have their plain and ordinary meaning when read in light ofthe specification, and includes, for example, substances obtained from aplant and used as an additive. Botanical substances may be extractedfrom plants or plant parts thereof (for example, flowers, seeds, leavesand roots). Some botanical substances may have antioxidants. In someembodiments, botanicals are selected from a group consisting of AloeBarbadensis Leaf Juice, Persea gratissima (Avocado) Oil, Rosmarinusofficinalis (Rosemary) Leaf Oil, Rosa canina (Rose Hip) Fruit Oil,Butyrospermum parkii (Shea) Butter, Paeonia albiflora (Peony) RootExtract, Phenyl t-Butylnitrone (Spin Trap), Camellia sinensis (WhiteTea) Leaf Extract, Lavandula angustifolia (Lavender) Oil, Santalumaustrocaledonicum (Sandalwood) Wood Oil and Hamamelis virginiana (WitchHazel).

Accordingly, embodiments provided herein relate to a composition thatmaintains or that is capable of maintaining a biologic, such as plateletrich plasma (PRP), or a component thereof, fibroblast cells, adiposetissue, adipose derived stromal vascular function (SVF), nanofat,lipoaspirate components, bone marrow derived mesenchymal stem cells,adipose derived stem cells, platelet derived exosomes, adipose derivedexosomes, alpha 2 macroglobulin (A2M), human platelet lysate, orisolated microparticles thereof.

As used herein, the term “maintain” refers to an ability of a compoundor agent to preserve, extend the life of, enhance, sustain, or otherwiseprolong a biologic over a given period of time in the compositionsdescribed herein. Maintain does not necessarily refer to completemaintenance, but may include partial maintenance, such as maintenance ofless than 100% of the biologic over a period of time. For example,maintain may include maintenance of an amount of at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% maintenance of the biologic over a period of time.The period of time may range in length from at least 1 day to at least500 days, for example, 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 30, 60, 90, 120,150, 180, 210, 240, 270, 300, 330, 360, 365, 400, 450, or 500 days, orfor a period of time within a range defined by any two of theaforementioned periods of time.

In some embodiments, the base composition includes a cell nutrient; abiological buffer; a viscosity modifying agent; a thymol; and ananti-microbial. As used herein, a cell nutrient includes, for examplefetal bovine serum (FBS), glucose, human platelet lysate, bovine serumalbumin, fibroblast growth supplement, vitamins, trace elements,amniotic cell culture supplements, or lipo-polysaccharides, or othercell supplements that are used to maintain a cell (such as afibroblast), a cell fragment (such as a platelet), or intracellular orextracellular components thereof (such as cytokines within a cell orcell fragment). As used herein, a biological buffer includes, forexample, any buffer of sodium, potassium, magnesium, calcium, alphahydroxy acid, beta hydroxy acid, polyhydroxy acid, hyaluronic acid,and/or carboxylic acid, or a cell culture buffering agent, or aderivative or any combination thereof. In some embodiments, thebiological buffer is sodium bicarbonate, sodium gluconate, sodiumchloride, sodium citrate, sodium acetate, potassium phosphate, potassiumchloride, and/or magnesium chloride. In some embodiments, the biologicalbuffer preserves the biologic at about or above physiological pH. Insome embodiments, the biological buffer comprises a cosmetic pHadjuster, including, for example, sodium hydroxide, citric acid, aceticacid, ascorbic acid, benzoic acid, malic acid, formic acid, fumaricacid, hydrochloric acid, glycolic acid, lactic acid, magnesiumhydroxide, maleic acid, malonic acid, nitric acid, sodium carbonate,azelaic acid, glucosamine, glycinate, propane derivative, proponal,sodium phosphate, sulfuric acid, triethanolamine, ammonium, lithiumhydroxide, magnesium carbonate, oxalic acid, phosphoric acid, tartaricacid, glyoxylic acid, imidazole, lactobionic acid, dibenzothiophene,glutaric acid, guanidine carbonate, galacturonic acid, calciumhydroxide, succinic acid, strontium hydroxide, or derivatives thereof,or any combination thereof.

In some embodiments, the biological buffer includes a cell culturebuffer, including, for example, MES (2-(N-Morpholino)ethanesulfonicacid, 4-Morpholineethanesulfonic acid), BIS-TRIS, ADA(N-(2-Acetamido)iminodiacetic acid), PIPES(Piperazine-N,N′-bis(2-ethanesulfonic acid), ACES, MOPSO(3-morpholinopropanesulfonic acid), BES(N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), MOPS(3-Morpholinopropane-1-sulfonic acid, 3-(N-Morpholino)propanesulfonicacid and 3-Morpholinopropanesulfonic acid), TES(2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonicacid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)),DIPSO (3-(N,N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid,N,N-Bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), TRISbase, Tricine (N-[Tris(hydroxymethyl)methyl]glycine), Gly-Gly(Glycyl-glycine), HEPPS (4-(2-Hydroxyethyl)-1-piperazinepropanesulfonicacid, 4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid,N-(2-Hydroxyethyl)piperazine-N′-(3-propanesulfonic acid)), Bicine(N,N-Bis(2-hydroxyethyl)glycine), TAPS(N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid,[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid),AMPD (2-Amino-2-methyl-1,3-propanediol, Ammediol), AMPSO(N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid),CHES (2-(Cyclohexylamino)ethanesulfonic acid), CAPSO(3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, CAPSO Free Acid),AMP (0-Aminoisobutyl alcohol, 2-Amino-2-methyl-1-propanol), or CAPS(3-(Cyclohexylamino)-1-propanesulfonic acid), or any derivative thereof,or any combination thereof.

A base composition for preserving, extending the life of, enhancing,sustaining, maintaining, or otherwise prolonging a biologic as providedherein may include a cell culture reagent, a buffering agent, aviscosity modifying agent, a thymol, or an anti-microbial agent, or anycombination thereof.

As used herein, a cell culture reagent may include any culture reagentthat is capable of maintaining a composition that includes biologicalsubstances, and can include any media as disclosed herein, including,for example, DMEM, IMDM, RPMI 1640, MEM, OPti-MEM medium, DMEM/F-12,GluaMax, Advanced media, Recovery freezing medium, F10 Nutrient mixture,Ham's F12 Nutrient mixture, Media 199, Opti-MEM, SensiCell Media forSensitive cells, BME, Stem cell media (Essential 8™, StemPro™, MSC SFMmedia), EMD-Millipore (Cellvento™, Cellvento™ BHK cell culture medium,Cellvento™ CHO cell culture media platform, Customized Cell CultureMedia, Classical Cell Culture Media), Fisher Scientific (Corning™Cellgro™ Minimal Essential Medium Eagle without Glutamine, Corning™Cellgro™ DMEM with L-glucose and sodium pyruvate), Stemcell™Technologies (mTESR™ 1, MethoCult™ H4034 Optimim, BrainPhys™), LifeTechnologies, Invitrogen™ (RPMI 1640 medium) and GE Healthcare (AciCHOP).

As used herein, a thymol refers to 2-isopropyl-5-methylphenol, and is anatural monoterpenoid phenol derivative of cymene, C₁₀H₁₄O, isomericwith carvacrol, found in oil of thyme, and extracted from Thymusvulgaris (common thyme) and various other kinds of plants as a whitecrystalline substance of a pleasant aromatic odor and strong antisepticproperties. Thymol may include, for example, a thymol isomer, a cresol,or O-cymen-5-ol.

As used herein, an anti-microbial agent, refers to an agent that hasantimicrobial properties. An anti-microbial agent may include, forexample, any agent known or discoverable for its ability to killmicrobes, including partial or complete antimicrobial properties. Ananti-microbial agent may include, for example, one or more of Loniceracaprifolium (honeysuckle) flower extract or Lonicera japonica(honeysuckle) flower extract, or a combination thereof.

In some embodiments, the base composition provided herein includes acell nutrient, a biological buffer, a viscosity modifying agent, and abotanical extract. As used herein, a botanical extract has its ordinarymeaning in light of the specification, and refers to a composition froma plant source (a botanical). A botanical extract can include, forexample, an aqueous extract, wherein the botanical extract is obtainedby soaking the botanical in a solvent to extract a portion of thebotanical into the solvent. In some embodiments, the extract is analcohol extract, such as a methanolic extract or an ethanolic extract(for example, a botanical extract obtained by extracting with a methanolmixture or with an ethanol mixture). In some embodiments, a botanicalextract is an aqueous ferment extract, such that the botanical is soakedin a solvent that includes a fermenting agent, such as a probiotic. Insome embodiments, the botanical extract includes a cosmetic compound. Insome embodiments, the botanical extract exhibits antimicrobialproperties, including antibacterial, antifungal, antiviral, orantiparasitic properties.

In some embodiments, a botanical extract is obtained from Cocos nuciferafruit, radish root, Sambucus nigra fruit, Ribes nigrum fruit, Populustremuloides bark, Wasabia japonica root, Zingiber officinale root,Allium sativum bulb, wasabi, honeysuckle, cedar wood, aspen bark, willowbark, Brahmi (Bacopa monnieri) extract, citrus extract Camellia sinensis(green tea), grapes, pomegranate, Echinacea, Centella asiatica,Elderflower, Irish moss, Mallow, soap bark, Yucca, Clary sage, oregano,thyme, curcumin compounds, resveratrol (polyphenolic compound fromgrape, berries, etc.) vetivert, chamomile, rosemary, aloe, nettle,Centella asiatica, Ginkgo biloba, betula, witch hazel, green tea, whitetea, grape skin, grape seed, resveratrol grapefruit, grapefruit seed,grapefruit peel, citrus fruits (other than grapefruit extract) bilberry,blueberry, Ginkgo biloba, soy isoflavones, soy extract, fermented soyprotein, black cohosh, St. John's wort, echinacea, chamomile, rosemary,aloe extract and juice, nettle, coconut fruit, or Centella asiatica, orcombinations thereof. The botanical utilized to obtain the botanicalextract may be obtained from any of the plant parts including theleaves, pulp, seeds, or stems, fruit and fruit seeds, as well as thewhole plant.

As used herein, “probiotics” has its ordinary meaning as understood inlight of the specification, and refers to live microorganisms thatconfer health benefits when consumed. In some embodiments, a probioticincludes Lactobacillus, Leuconostoc, Bacillus, Saccharomyces,Pediococcus, Weissella, Bifidobacterium, Streptococcus, Lactococcus,Gluconacetobacter, Zygosaccharomyces, Acetobacter, or Gluconobacter, orany derivative, ferment, or ferment filtrate thereof, or any combinationthereof.

In some embodiments, the cosmetic compound includes a salicylate, aphenolic compound, or a phytonutrient. In some embodiments, thesalicylate is derived from an aspen bark isolate, willow bark isolate,or other botanical having salicylate.

As used herein, a “phenolic compound” has its ordinary meaning asunderstood in light of the specification, and refers to a group of smallmolecules having at least one phenol unit. Phenolic compounds caninclude, for example, phenolic acids, flavonoids, tannins, coumarins,lignans, quinones, stilbenes, or curcuminoids. Examples of phenolicacids include, for example, hydroxybenzoic acids (including, forexample, gallic acid, gentisic acid, 4-hydroxybenzoic acid,protocatechuic acid, β-resorcylic acid, salicylic acid, syringic acid,or vanillic acid) or hydroxycinnamic acids (including, for example,chlorogenic acid, trans-cinnamic acid, caffeic acid, p-coumaric acid,ferulic acid, isoferulic acid, or sinapic acid), or derivatives oranalogues thereof. Examples of flavonoids include, for example,flavan-3-ols (including, for example, (+)-catechin, (−)-epicatechin,(−)-gallocatechin gallate, (−)-epigallocatechin gallate, (−)-epicatechingallate, (−)gallocatechin, or (−)-epigallocatechin), flavonols(including, for example, datiscetin, fisetin, flavonol, galangin,hyperoside, isorhametin, kaempferol, morin, myricetin, myricitrin,quercetagetin, quercetin, quercitrin, robinetin, rutin, or tamarixetin),flavones (including, for example, apigenin, aposide, balcalcin,balcalin, chrysin, chrysoeriol, diosmetin, gardenin A, genkwanin,lueolin, vitexin, isovitexin, sinensetin, tangeretin, or wogonoside),isoflavones (including, for example, biochanin A, daidzein, daidzin,formononetin, genistin, genistein, glycitin, glycitein, or pucrarin),flavanones (including, for example, dihydromyricetin, eriodictyol,hesperetin, hesperidin, neohesperidin, liquiritigenin, liquiritin,naringenin naringin, narinatin, or (+)-taxifolin), or anthocyanidins(including, for example, cyaniding, delphinidin, malvidin, pelargonidin,peonidin, petunidin, or propelargonidin) or analogues or derivativesthereof. Examples of tannins include, for example, hydrolyzable tannins(including, for example, gallotannins or ellagitannins) or condensedtannins (including, for example, proanthocyanidins orleucoanthocyanidins) or analogues or derivatives thereof. Examples ofcoumarins include, for example, simple coumarins, furanocoumarins,pyranocoumarins, isocoumarins, or analogues or derivatives thereof.Examples of lignans include, for example, lignanolides,cyclolignanolides, bisepoxylignans, neolignans, or analogues orderivatives thereof. Examples of quinones include, for example,anthraquinones, phenanthraquinones, napthoquinones, benzoquinones, oranalogues or derivatives thereof. Examples of stilbenes include, forexample, isorhapontigenin, oxyresveratrol, piceid, piceatannol,pinostilbene, pterostilbene, resveratrol, or analogues or derivativesthereof. Examples of curcuminoids include, for example, curcumin orginerol analogues, or analogues or derivatives thereof. Other phenoliccompounds may include, for example, chalcone derivatives,isoliquiritigenin, butein, phloretin, phenolic alkaloids, phenolicterpenoids, m-benzo-triphenol derivatives, or cresol, or derivatives oranalogues thereof. In some embodiments, the phenolic compound is athymol or analogue or derivative thereof, such as O-cymen-5-ol.

As used herein, “phytonutrient” has its ordinary meaning as understoodin light of the specification, and refers to a plant derived substanceassociated with positive health effects. A phytonutrient may broadlyinclude natural materials having antimicrobial properties, and furtherexhibit antioxidant and/or anti-inflammatory properties. Phytonutrientsmay be derived from a variety of botanicals, such as from Sambucu nigrafruit extract, Populous termuloides bark extract, Ribes nigurm fruitextract, other botanicals. A phytonutrient may include, for examplebetalain, indole, organosulfide, phenol, terpene, triterpene,carotenoid, curcuminoid, flavonoids, glucosinolate, isothiocyanate,hydroxycinnamic acid, lignan, lipid, stilbene, sulphide, tocopherol,lutein, zeanthin, isoflavone, flavonoid, coumestna, lycopene, ellagicacid, caffeoylquinic acid, hydroxybenzoic acid, hesperetin, flavonol,terpenoid, phthalide, flavonol, allicin quercetin, sulphide,anthocyanin, resveratrol, and anthoxanthin. In some embodiments, thephytonutrient is a pigmented phytonutrient, such as anthocyanin, lutein,zeaxanthin, lycopene, carotenoids, and/or anthoxanthin.

Some embodiments include any combination of the aforementioned botanicalextracts with any of the aforementioned probiotics and/or cosmeticcompounds. Non-limiting examples of such combinations include, forexample; Lactobacillus and Cocos Nucifera fruit extract; Leuconostoc andRadish root ferment filtrate; Lactobacillus ferment; Bacillus fermentand Saccharomyces ferment filtrate; Leuconostoc, radish root fermentfiltrate, Lactobacillus, and Cocos nucifera fruit extract; Lactobacillusferment, Lactobacillus, and Cocos Nucifera fruit extract; and hexyleneglycol, caprylyl glycol, Wasabia japonica root extract, Zingiberofficinale root extract, and Allium sativum bulb extract.

In some embodiments, the base composition includes a cell nutrient, abiological buffer, a polymer, a thymol, and an antimicrobial. As usedherein a “polymer” has its ordinary meaning as understood in light ofthe specification, and refers to a polymeric compound prepared bypolymerizing monomers, whether of the same or a different type. Thegeneric term polymer thus embraces the term homopolymer (employed torefer to polymers prepared from only one type of monomer, with theunderstanding that trace amounts of impurities can be incorporated intothe polymer structure), and the term interpolymer, which are polymersprepared by the polymerization of at least two different types ofmonomers. The generic term interpolymer thus includes copolymers(employed to refer to polymers prepared from two different types ofmonomers), and polymers prepared from more than two different types ofmonomers. The term polymer includes trace amounts of impurities, forexample catalyst residue, that may be incorporated into and/or withinthe polymer. In some embodiments, the polymer includes copolymer or acrosspolymer, or a derivative thereof. In some embodiments, the polymerincludes an acrylate crosspolymer, an acrylate copolymer, or a siliconecrosspolymer, or derivatives thereof, such as polyacrylatecrosspolymer-6, dimethicone crosspolymer, hydroxyethyl acrylate/sodiumacryloyldimethyl taurate copolymer, or derivatives thereof. In someembodiments, the polymer includes a natural or a synthetic polymer, andmay include, for example, starch, xanthan gum, guar gum, carrageenan,alignates, polysaccharids, pectin, gelatin, agar, cellulose,polyacrylate, polyacrylamide, silicone or derivates or analoguesthereof.

In some embodiments, a base composition includes a cell nutrient, abiological buffer, a polymer, a thymol, an antimicrobial, a viscositymodifying agent, a phytonutrient, a phenolic compound, a salicylate, abotanical extract, or any combination of any of the aforementionedcompounds or agents.

In any of the embodiments described herein, the first and/or secondformulation includes a skin conditioning agent, an emulsion stabilizer,a humectant, a chelating agent, a biocide, a buffering agent, a biologicbuffering agent, an antioxidant, a pH adjuster, a viscosity modifyingagent, a sun protection agent, a cell culture reagent, an enzymeinhibitor, an activation inhibitor, a skin permeability enhancer, a skindelivery system, or a combination thereof.

In some embodiments, the base composition further includes a proteintransport inhibitor agent, including, for example, brefeldin A ormonensin, or a combination thereof.

In some embodiments, the first and/or second formulation includes a skinconditioning agent. A skin conditioning agent as used herein refers to asubstance that is used to maintain, improve, or enhance a skincondition, and may include, for example, water, Lonicera japonica(honeysuckle) flower extract, niacinamide, Lonicera caprifolium(honeysuckle) flower extract, Persea gratissima (avocado) oil, aloebarbadensis leaf juice, sodium hyaluronate (L), Althaea officinalis(marshmallow) root extract, caprylyl methicone, retinyl palmitate, amixture of palmitoyl tri- and hepta-peptides, ectoin, acetylglucosamine, panthenol, caprylyl glycol, caffeine, lactic acid/glycolicacid copolymer, allantoin or bisabolol, or combinations thereof.

In some embodiments, the first and/or second formulation includes anemulsion stabilizer. An emulsion stabilizer refers to a compound ormixture of compounds that helps maintain an emulsion. In someembodiments, the emulsion stabilizer includes, for example, polyacrylatecrosspolymer-6, an alcohol, cetyl alcohol, stearyl alcohol, acetic acid,carbomer, gum palmitic acid, polyethylene glycol, isostearic acid,stearic acid, or mixtures thereof.

In some embodiments, the first and/or second formulation includes ahumectant. A humectant as used herein refers to a compound or mixture ofcompounds that increase water content of the top layers of skin. Ahumectant may include, for example, glycerin, glucose (D), polyhydricalcohols such as glycerol, other polyalkylene glycols (e.g., dipropyleneglycol, polypropylene glycol, and polyethylene glycol) and derivativesthereof, alkylene polyols and their derivatives, sorbitol, hydroxysorbitol, hexylene glycol, 1,3-dibutylene glycol, 1,2,6-hexanetriol,ethoxylated glycerol, propoxylated glycerol, sodium hyaluronate, ormixtures thereof.

In some embodiments, the first and/or second formulation includes achelating agent. A chelating agent refers to a compound or mixture ofcompounds that stabilize compositions by preventing precipitation bybinding to a particular compound. A chelating agent may include, forexample, citric acid, sodium gluconate, EDTA (ethylenediaminetetraacetic acid), HEDTA (hydroxyethylenediamine triacetic acid), NTA(nitriolotriacetic acid), DTPA (diethylenetriaminepentaacetic acid),MGDA (methylglycinediacetic acid), HEIDA (2-hydroxyethyliminodiaceticacid), CDTA (trans-cyclohexane-1,2-diaminetetraacetic acid), EGTA(ethylene glycol-bis((3-aminoethyl ether)-N,N,N′,N′-tetraacetic acid),EDDA (ethylenediaminediacetic acid), propylene diamine tetraacetic acid(PDTA), ethylene diamine-N,N″-di(hydroxyphenylacetic) acid (EDDHA),ethylene diamine-N,N″-di-(hydroxy-methylphenyl acetic acid (EDDHMA), orsodium, potassium, and/or ammonium salts, or combinations thereof.

In some embodiments, the first and/or second formulation includes abiocide. A biocide is a compound or mixture of compounds for use informulations that are used to eliminate, remove, reduce, prevent, orprotect against harmful or unwanted organismal growth. A biocide mayinclude a cosmetic biocide, and may include, for example, ammoniumphenolsulfonate, benzalkonium chloride, benzethonium chloride,cetylpyridinium chloride, chlorophyllin-copper complex, chlorothymol,chloroxylenol, cloflucarban, dichloro-m-xylenol, methylbenzethoniumchloride, O-cymen-5-OL, phenol, sodium phenolsulfonate, triclocarban,triclosan, zinc phenolsulfonate, zinc ricinoleate, or mixtures thereof.

In some embodiments, the first and/or second formulation includes aviscosity modifier. A viscosity modifying agent is a compound or mixtureof compounds that modifies viscosity of a composition, and may eitherincrease or decrease the viscosity of a formulation, depending onwhether it is desirable to have a formulation with increased ordecreased viscosity. For example, a viscosity modifying agent caninclude any substance suitable to achieve a viscosity of about 1 toabout 10 centipoise. The viscosity modifier may include, for example,glycerol, glycerol derivatives, ethylene glycol, propylene glycol(dihydroxypropane), polyethylene glycol, water soluble polymer, naturalhydrocolloids (and derivatives), Acacia, tragacanth, alginic acid,carrageenan, locust bean gum, guar gum, xanthan gum, gum arabic,gelatin, cellulose, alginates, starches, honeys, hydrogels, chitosans,dextrans, gelatin, sugars (and derivatives), dextrose, fructose,polydextrose, polydextrans, saccharides, polysaccharides, semisynthetichydrocolloids (and derivatives), methylcellulose, hydroxyethyl starch(hetastarch), sodium carboxymethylcellulose, sodium chloride, potassiumchloride, magnesium chloride, hydroxyethylcellulose,hydroxypropylmethylcellulose, polyvinylpyrrolidone (PVP), synthetichydrocolloids (and derivatives), Polyvinyl alcohol (PVA) or Carbopol® orcombinations thereof.

In some embodiments, the first and/or second formulation includes abuffering agent. A buffering agent refers to an agent use to maintain pHof a solution near a chose pH value. Such agents may include acids,bases, or combinations thereof. Buffering agents may include biologicbuffering agents, and may include, for example, for example, phosphatesalts, boric acids or boric salts, magnesium chloride, potassiumchloride, glucose, sodium bicarbonate, sodium chloride, sodium acetate,sodium phosphate, fetal bovine serum (FBS),N-cyclohexyl-2-aminoethanesulfonic acid (CHES) or salts thereof,3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonicacid (TAPS) or salts thereof, 2-(N-morpholino)ethanesulfonic acid (MES)and salts thereof, piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES) andsalts thereof, N-(2-acetamido)-2-aminoethane-sulfonic acid (ACES) andsalts thereof, cholamine chloride, BES,2-[[1,3-dihydroxy-2-(hydroxymethyl)-propan-2-yl]amino]ethanesulfonicacid (TES) and salts thereof,2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) andsalts thereof, acetamidoglycine;N-(2-hydroxy-1,1-bis(hydroxyl-methyl)ethyl)glycine (tricine),glycinamide, 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine) and saltsthereof, propionate salts,3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]-amino]-2-hydroxy-propane-1-sulfonicacid (TAPSO) and salts thereof, 3-morpholinopropane-1-sulfonic acid(MOPS) and salts thereof, saline-sodium citrate (SSC) buffer,2-amino-2-hydroxymethyl-propane-1,3-diol (synonyms: TRIS, trisamine,THAM, tromethamine, trometamol, tromethane), citric acid or citratesalts (e.g. sodium citrate), trisodium phosphate, disodium hydrogenphosphate, sodium dihydrogen phosphate, tripotassium phosphate,dipotassium phosphate, monopotassium phosphate and/or any otherbuffering agent containing phosphate, or combinations thereof.

In some embodiments, the first and/or second formulation includes anantioxidant. An antioxidant refers to a compound or mixture of compoundsthat inhibit, suppress, or reduce oxidative damage to cells orbiomolecules by stabilizing free radicals before they can cause harmfulreactions. Antioxidants may include, for example, tocopheryl acetate(D-alpha), vitamins, vitamin cofactors, minerals, hormones, carotenoids,carotenoid terpenoids, non-carotenoid terpenoids, flavonoids, flavonoidpolyphenolics (e.g., bioflavonoids), flavonols, flavones, phenols,polyphenols, esters of phenols, esters of polyphenols, nonflavonoidphenolics, isothiocyanates, vitamin A, vitamin C, vitamin E, ubiquinone,mineral selenium, manganese, melatonin, a-carotene, β-carotene,lycopene, lutein, zeanthin, crypoxanthin, reservatol, eugenol,quercetin, catechin, gossypol, hesperetin, curcumin, ferulic acid,thymol, hydroxytyrosol, tumeric, thyme, olive oil, lipoic acid,glutathinone, gutamine, oxalic acid, tocopherol-derived compounds,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),ethylenediaminetetraacetic acid (EDTA), tert-butylhydroquinone, aceticacid, pectin, tocotrienol, tocopherol, coenzyme Q10, zeaxanthin,astaxanthin, canthaxantin, saponins, limonoids, kaempfedrol, myricetin,isorhamnetin, proanthocyanidins, quercetin, rutin, luteolin, apigenin,tangeritin, hesperetin, naringenin, erodictyol, flavan-3-ols (e.g.,anthocyanidins), gallocatechins, epicatechin and its gallate forms,epigallocatechin and its gallate forms (ECGC), theaflavin and itsgallate forms, thearubigins, isoflavone, phytoestrogens, genistein,daidzein, glycitein, anythocyanins, cyaniding, delphinidin, malvidin,pelargonidin, peonidin, petunidin, ellagic acid, gallic acid, salicylicacid, rosmarinic acid, cinnamic acid and its derivatives (e.g., ferulicacid), chlorogenic acid, chicoric acid, gallotannins, ellagitannins,anthoxanthins, betacyanins and other plant pigments, silymarin, citricacid, lignan, antinutrients, bilirubin, uric acid, R-a-lipoic acid,N-acetylcysteine, emblicanin, apple extract, apple skin extract(applephenon), rooibos extract red, rooibos extract, green, hawthornberry extract, red raspberry extract, green coffee antioxidant (GCA),aronia extract 20%, grape seed extract (VinOseed), cocoa extract, hopsextract, mangosteen extract, mangosteen hull extract, cranberry extract,pomegranate extract, pomegranate hull extract, pomegranate seed extract,hawthorn berry extract, pomella pomegranate extract, cinnamon barkextract, grape skin extract, bilberry extract, pine bark extract,pycnogenol, elderberry extract, mulberry root extract, wolfberry (gogi)extract, blackberry extract, blueberry extract, blueberry leaf extract,raspberry extract, turmeric extract, citrus bioflavonoids, blackcurrant, ginger, acai powder, green coffee bean extract, green teaextract, and phytic acid, or synthetic antioxidant such as butylatedhydroxytolune or butylated hydroxyanisole, or combinations thereof.

In some embodiments, the first and/or second formulation includes a pHadjuster. A pH adjuster refers to a compound or mixture of compoundsthat can change the pH of a composition. Exemplary pH adjusters includeacids, bases, buffers, phosphates (such as potassium phosphate) orcombinations thereof.

In some embodiments, the first and/or second formulation includes a sunprotection agent. A sun protection agent refers to a compound or mixtureof compounds that provide protection to skin against damaging effects ofthe sun. In some embodiments, the sun protection agent providesprotection against ultraviolet radiation. A sun protection agent mayinclude, for example, petrolatum, metal oxides, zinc oxide, titaniumdioxide, para-aminobenzoic acid, PABA esters (glyceryl PABA,amyldimethyl PABA and octyldimethyl PABA), butyl PABA, ethyl PABA, ethyldihydroxypropyl PABA, benzophenones (oxybenzone, sulisobenzone,benzophenone, and benzophenone-1 through 12), cinnamates (octylmethoxycinnamate (octinoxate), isoamyl p-methoxycinnamate, octylmethoxycinnamate, cinoxate, diisopropyl methyl cinnamate, DEA-methoxycinnamate,ethyl diisopropylcinnamate, glyceryl octanoate dimethoxycinnamate andethyl methoxycinnamate), cinnamate esters, salicylates (homomethylsalicylate, benzyl salicylate, glycol salicylate, isopropylbenzylsalicylate, etc.), anthranilates, ethyl urocanate, homosalate,octisalate, dibenzoylmethane derivatives (e.g., avobenzone),octocrylene, octyl triazone, digalloyl trioleate, glycerylaminobenzoate, lawsone with dihydroxyacetone, ethylhexyl triazone,dioctyl butamido triazone, benzylidene malonate polysiloxane,terephthalylidene dicamphor sulfonic acid, disodium phenyldibenzimidazole tetrasulfonate, diethylamino hydroxybenzoyl hexylbenzoate, bis diethylamino hydroxybenzoyl benzoate, bisbenzoxazoylphenyl ethylhexylimino triazine, drometrizole trisiloxane,methylene bis-benzotriazolyl tetramethylbutylphenol, andbis-ethylhexyloxyphenol methoxyphenyltriazine, 4-methylbenzylidenecamphor, isopentyl 4-methoxycinnamate, kaolin, talc, or derivatives oranalogues thereof, or combinations thereof.

In some embodiments, the first and/or second formulation includes a cellculture reagent. A cell culture reagent is a compound or mixture ofcompounds used in cell culture for proliferation, maintenance, orinduction of a cell culture. Cell culture reagents may include, forexample, cell culture media (including the growth media describedherein), cell culture serum, cell culture additives, media supplements,feeder cells, or combinations thereof.

In some embodiments, the first and/or second formulation includes anenzyme inhibitor. An enzyme inhibitor is a compound or mixture ofcompounds that inhibits or reduces function and/or activity of anenzyme. An inhibitor may prevent a substrate from entering the activesite of the enzyme and/or prevent the enzyme from catalyzing a chemicalreaction. Enzyme inhibitors may be reversible (non-covalently bound tothe enzyme) or irreversible (covalently bound to the enzyme).

In some embodiments, the first and/or second formulation includes anactivation inhibitor. An activation inhibitor is a compound or mixtureof compounds that prevents or reduces activation of a biologic. Abiologic may undergo activation, thereby resulting in subsequent lysisand death. For example, platelet activation results in platelet lysisand death. Activation of a biologic is desired to be controlled, andpreferably at the time of or after application of a formulation to asubject. An activation inhibitor is capable of reducing, minimizing, orpreventing activation of the biologic during storage or after theformulation is prepared but prior to application of the formulation. Insome embodiments, the activation inhibitor prevents activation for aperiod of greater than 15, 30, 45, 60, 75, 90, 105, or 120 days, orgreater. An activation inhibitor may include, for example, an inhibitoror chelator of thrombin, calcium chloride, batroxobin, or collagen.

In some embodiments, the first and/or second formulation includes a skinpermeability enhancer. A permeability enhancer increases or enhances anability of a formulation or compounds within a formulation to penetrateor absorb into skin. A kin permeability enhancer may include, forexample, magainin, short synthetic peptide (ACSSSPSKHCG), biotinylatedhepta-D-arginine, sulphoxides (such as dimethylsulphoxide, DMSO), Azones(e.g. laurocapram), pyrrolidones (for example 2-pyrrolidone, 2P),alcohols and alkanols (ethanol or decanol), glycols (for examplepropylene glycol), surfactants, or terpenes, or combinations thereof.

In some embodiments, the first and/or second formulation includes a skindelivery system. A skin delivery system refers to a combination ofcompounds that enhance delivery of a formulation to skin, and mayinclude, for example, emulsions (such as micro- and nano-encapsulationemulsions), vesicles, liposomes, solid-lipid nanoparticles (SLN),nanostructured lipid carriers (NLC), phytosomes, transferosomes,nanocrystals, cubosomes, or fulvate fraction liposomes, or anycombination thereof.

“Foam” has its plain and ordinary meaning when read in light of thespecification, and includes, for example, a substance that is formed bytrapping pockets of gas in a liquid or solid. In some alternativeformulations herein, a formulation is provided as a foam to be absorbedinto the skin.

“Batroxobin,” has its plain and ordinary meaning when read in light ofthe specification, and includes, for example, snake venom produced byBothrops atrox and Bothrops moojeni, venomous species of pit viper.Batroxobin may act as a hemotoxin which acts as a serine proteaseclosely related to thrombin and has been the subject of many medicalstudies as a replacement of thrombin.

This disclosure provides for a method of treating for a skin disorder ortreating signs of again in a subject in need. This disclosure alsodescribes formulations for treating a skin disorder and signs of aging.A kit comprising formulations is also contemplated.

Methods of Treating Skin of a Subject

In some embodiments, a method of preparing a formulation for treatmentis provided. A method of making a topical formulation for treatment fora subject is also provided. Previous methods of preparing mammaliancells involve the use of media, wherein the media contains fetal bovineserum. As shown in exemplary embodiments herein, formulations comprisinga biologic, such as cells, platelet rich plasma, human platelet lysateor combinations thereof, in combination with a base composition may leadto a surprising increase in the proliferation or longevity of thebiologic with a shorter doubling time of cells, which is more costeffective. In some embodiments, the biologic also exhibit an increase inthe expression of the protein collagen, which is known to improve theappearance of the skin. Additionally, the increase in proliferation orlongevity of the biologic can lead to a larger amount of biologics to beused for treatment and to treat a larger surface area of skin on asubject in need. Human platelet lysate can be prepared for use, which isknown to those skilled in the art. Furthermore, commercially availablehuman platelet lysate can be used, for example, such as HPL preparedcommercially by Mill Creek Life Sciences, Compass Biomedical, Inc., CookRegentec, Macopharama SA, iBiologics, PL bioscience GmbH and TrinovaBiochem GmbH. Product lines can include but are not limited to PLTMax,PLUS™, Stemulate, Human Platelet Lysate, XcytePlus, PL_(SOLUTION),PL_(MATRIX) and CRUX RUFA Media supplements. Furthermore, platelet richplasma and platelet lysate may also be prepared from the subject fortheir own treatment. Activation of the platelets in the platelet richplasma is also contemplated, as this would allow degranulation or theplatelets, allowing growth factors to be released and absorbed into theskin.

Human platelet lysate can also be prepared for use in the embodimentsdescribed herein, and their preparation is known to those skilled in theart. The preparation of human platelet lysate is described inSchallmoser et al. (J Vis Exp. 2009 Oct. 30; (32), Fekete et al.(Cytotherapy. 2012 May; 14(5): 540-554) and Shih et al. (NewBiotechnology Volume 32, Issue 1, 25 Jan. 2015, Pages 199-211) (each ofthese references, and all references cited herein, are hereby expresslyincluded by reference in their entireties). Human platelet lysate canalso be produced by use of a commercially available platelet lysatepreparation kit, such as ones by Macopharma. The human platelet lysatecan be comprised from but not limited to platelet rich plasma (PRP),pooled platelets from humans and cultured megakaryocytes from stem cellexpansion technology.

As used herein, human platelet lysate can be a substitute supplement forfetal bovine serum in experimental as well as in clinical cell cultures.The human platelet lysate is obtained from human blood platelets afterseveral freeze/thaw cycles, which case the platelets to lyse and releasea large quantity of growth factors for cell expansion. In someembodiments, the media in which the cells are growing comprise 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% volume/volumehuman platelet lysate in the medium, or any amount in between any twoaforementioned values. In some embodiments, whole blood from a subjecthaving skin damage is obtained and centrifuged. The centrifugation leadsto the separation of the blood into fractions of platelet rich plasmafrom platelet poor plasma and red blood cells. The platelet rich plasmais then separated. The platelet rich plasma may be added to a basecomposition, wherein the base compositions is a topical cosmetic base, agel, a serum, a lotion, an ointment, a spray, an aerosol, a powder, asolution, a liquid, a foam, a salve, a paste, or a cream or combinationthereof. In some embodiments, the platelet rich plasma is subjected to afreeze thaw cycle to break up the platelets, thus forming plateletlysate.

The first formulation may be stored in a temperature controlled unit,such as a refrigerator, prior to use to preserve the PRP of theformulation. The formulation can be stored in a refrigerator for aperiod of more than 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 120days, or an amount of days within a range defined by any two of theaforementioned values. The first formulation may be stored at 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30° C., or at atemperature within a range defined by any two of the aforementionedvalues.

In some embodiments, the first formulation comprises a base compositionin which the cells are mixed into thereby producing the firstformulation. In some embodiments, the base composition of the firstformulation comprises aqua (water), Lonicera japonica (honeysuckle)flower extract, glycerin, niacinamide, polyacrylate crosspolymer-6,Lonicera caprifolium (honeysuckle) flower extract, Persea gratissima(avocado) oil, Aloe barbadensis leaf juice, sodium gluconate, sodiumhyaluronate (L), Althaea officinalis (marshmallow) root extract,O-cymen-5-OL, sodium hydroxide, glucose (D), sodium chloride, sodiumcitrate, sodium acetate, sodium bicarbonate, tocopheryl acetate(D-alpha), potassium phosphate, potassium chloride, magnesium chloride,phenoxyethanol, Anthemis nobilis (chamomile) flower oil, Rosmarinusofficinalis (rosemary) leaf oil, Thymus vulgaris (thyme) flower/leafoil, benzyl alcohol, caprylyl glycol, caprylhydroxamic acid, orcombinations thereof. In some embodiments, any of the aforementionedcomponents are present in an amount from about 0.001 wt % to about 99%wt %, including 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02, 0.03,0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%,or an amount within a range defined by any two of the aforementionedvalues.

In some embodiments, a method of treating skin on a subject sufferingfrom skin damage and/or signs of aging is provided. The method comprisesobtaining whole blood from a subject having skin damage, centrifugingthe blood to separate platelet rich plasma from platelet poor plasma andred blood cells, collecting the platelet rich plasma (PRP), adding abase composition, wherein the base composition is a topical cosmeticbase, a gel, a serum, a lotion, an ointment, a spray, an aerosol, apowder, a solution, a liquid, a foam, a salve, a paste, or a cream orcombination thereof to the platelet rich plasma to form platelet richplasma admixed solution thereby forming a first topical formulation,providing the first topical formulation to the subject for treatment ofthe skin damage for a 90 day time period, wherein the providingcomprises instructions for the subject to topically apply the firsttopical formulation to an area of the skin and skin damage 1, 2, 3 or 4times a day, for up to 30, 60, 90 or 120 days or any number of days inbetween a range defined by any two aforementioned values, activating theplatelet rich plasma. In some embodiments, activating the PRP comprisestime, chemical, or temperature activation. Time activation may occur,for example, by providing sufficient time for the biologic, such as PRPto be activated. Chemical activation may occur, for example, byproviding a chemical that induces activation, such as by the use of acell stimulation cocktail. A cell stimulation cocktail may include, forexample, phorbol 12-myristate 13-acetate (PMA), ionomycin, or otherchemical agents. Such chemical agents may include, for example, calcium,CaCl₂), Calcium carbonate, thrombin, autologous thrombin, bovinethrombin, collagen type I, magnesium, sodium, components of snake venom,or any other agent capable of activating platelets or allowing plateletsto degranulate, or any combinations thereof. Thus, chemical activationmay occur, in some embodiments, by providing a second topicalformulation having activator agents to the subject. In some embodiments,the providing comprises applying the second topical formulation over thearea of the skin and skin damage that has received the first topicalformulation, wherein treating the skin damage and signs of aging isselected from the group consisting of diminishing pore size, diminishingbacteria damage, diminishing red areas, reducing skin damage caused bysun, and maintaining or improving skin tone. The first formulation maynot be applied to the ears, eyes, mouth area or other body orifices.

Temperature activation may occur, for example, by changing thetemperature of the base composition. In some embodiments, a change intemperature induces stress on the biologic, such as on PRP or platelets,thereby degranulating the biologic, resulting in activation of thebiologic. Thus, in some embodiments, temperature activation occurs bydecreasing the temperature or by increasing the temperature of the basecomposition. In one embodiment, the base composition is prepared havinga biologic, and is stored at refrigeration, such as a temperature ofless than about 10° C., such as 10° C., 9° C., 8° C., 7° C., 6° C., 5°C., 4° C., 3° C., 2° C., or 1° C. The refrigerated base composition isstored for a period of time, and is then applied to skin of a subject,such as to a face of a subject. Application of the base composition tothe skin of the subject causes the temperature of the base compositionto increase from refrigerated temperature to greater than roomtemperature, whereupon the biologic is activated due to the increase intemperature. In some embodiments, a kit is provided, wherein the kitincludes a base composition and a means for refrigeration of the basecomposition, such as a refrigerator or cooling device. The kit may beused by adding a biologic to the base composition, and placing thecomposition in the refrigerator or cooling device, where the compositionis stored until application. Upon application of the base composition ora portion thereof, the base composition increases in temperature,resulting in activation of the biologic, such as activation of PRP,platelets, or components thereof.

In some embodiments, the methods include use of a temperaturedifferential to activate a biologic, such as activation of PRP,platelets, or components thereof. For example, a volume of the basecomposition at room temperature may be topically applied to a user, forexample to a hand of a user, and mechanical force may be applied toapply the base composition onto the skin. The resulting change intemperature of the base composition, due to the surface temperature ofthe skin and/or the application of mechanical force, activates thebiologic. Without wishing to be limited to mechanistic actions, suchactivation of a biologic results in release of the biologic contents,enabling the ability of the biologic contents to penetrate skin of thesubject, as described herein.

Mechanical activation can include agitation, such as rubbing, massaging,spreading, or otherwise topically applying the compositions. Other meansof mechanical force may include use of a mechanical device, such as, forexample, microdermabrasion, oscillating fibers (Clarisonic), suction,microneedling, roller, or other mechanical cleansing devices. Mechanicalforce may also be complemented with ultrasound, radiofrequency,electromagnetic energy, or other sources of radiation to increasetemperature of the composition during application to the skin.

In some embodiments, contacting the base composition with the biologic,preserves, extends the life of, enhances, maintains, sustains, orotherwise prolongs the biologic, such as cells, proteins, antibodies,blood, serum, plasma, or components thereof, or other biologicalextracts.

In some embodiments, the second formulation is a gel, liquid, cream orlotion. In some embodiments, the second formulation is a liquid andwherein the liquid is applied as a spray such as an aerosol spray orpump spray. In some embodiments, the second formulation comprises aplatelet rich plasma activator. In some embodiments, the secondformulation further includes Batroxobin, epinephrine and/or thrombin. Insome embodiments, the second formulation comprises calcium, magnesium,sodium, components of snake venom or combinations thereof. In someembodiments, the snake venom comprises oxydoreductases, transferases,hydrolases, or lyases or a combination thereof. The calcium may beprovided as calcium chloride or calcium carbonate, for example. In someembodiments, the snake venom comprises Batroxobin. In some embodiments,the snake venom comprises dehydrogenase lactate, L-amino-acid oxidase,Catalase, Alanine amino transferase, Phospholipase A2,Lysophospholipase, Acetylcholinesterase, Alkaline phosphatase, Acidphosphatase, 5′-Nucleotidase, Phosphodiesterase, Deoxyribonuclease,Ribonuclease 1, Adenosine triphosphatase, Amylase, Hyaluronidase,NAD-Nucleotidase, Kininogenase, Factor-X activator, Heparinase,α-Fibrinogenase, β-Fibrinogenase, Fibrinolytic enzyme, Prothrombinactivator, Collagenase, Elastase or Glucosamine ammonium lyase orcombinations thereof. In some embodiments, the snake venom comprisesα-neurotoxins, β-neurotoxins, κ-Toxins, Dendrotoxins, Cardiotoxins,Myotoxins, Sarafotoxins or Hemorrhagins, or combinations thereof. Insome embodiments the snake venom comprises α-Bungarotoxin, α-toxin,erabutoxin, cobratoxin, Notexin, ammodytoxin, β-Bungarotoxin, crotoxin,taipoxin, κ-Toxin, Dendrotoxin, toxins I and K, y-Toxin, cardiotoxin,cytotoxin, Myotoxin-a, crotamine, Sarafotoxin a, Sarafotoxin b,Sarafotoxin c, Phospholipase A2, mucrotoxin A, hemorrhagic toxins, HT1or HT2 or combinations thereof. In some embodiments, the secondformulation further includes Batroxobin, epinephrine and/or thrombin.The second formulation may not be applied to the ears, eyes, mouth areaor other orifices. In some embodiments, wherein the second formulationcomprises thrombin, the second formulation may not be applied to theears, eyes, mouth area or other orifices. In some embodiments, whereinthe second formulation includes thrombin, the second formulation may notbe applied to the ears, eyes, mouth area or other body orifices. In someembodiments, the snake venom comprises Batroxobin. In some embodiments,the second formulation comprises 10% calcium (e.g. calcium ion, calciumchloride calcium carbonate, calcium gluconate). In some embodiments, thesecond formulation comprises 15%-20% ethanol, 1:4 ratio autologousthrombin to PRP, 10% calcium chloride w/10,000 units of bovine thrombin,batroxobin, chitosan, or any combination thereof.

In some embodiments, the biologic, such as PRP is activated by a changein temperature, such as by an increase or decrease in temperature. Forexample, in some embodiments, the first topical formulation (the basecomposition) having a biologic is stored at a refrigerated temperature,such as at a temperature of less than about 10° C., such as at atemperature of 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° C.,2° C., or 1° C. Storage of the base composition at low temperature mayserve to prevent activation of PRP. Upon application of the basecomposition to skin of a subject, for example, to the face of thesubject, the base composition increases in temperature, for example, toa temperature of greater than 10° C., including to room temperature orto a temperature greater than room temperature, such as bodytemperature. The increase in temperature may serve to activate PRP, suchthat the PRP is activated only upon application of the composition tothe subject.

In some embodiments, the subject is also suffering from a skin disorder.In some embodiments, the skin damage and signs of aging are caused bythe skin disorder. In some embodiments, the skin damage and signs ofaging are caused by smoking, alcohol, diet, extreme temperatures,chemicals, stress, lack of sleep, poor diet, poor immune system or acombination thereof. In some embodiments, the skin disorder is selectedfrom a group consisting of stretch marks (striae), psoriasis, skincancer, acne, alopecia, carbuncles, dermatitis, eczema, atopicdermatitis, contact dermatitis, seborrheic dermatitis, cradle cap,perioral dermatitis, shingles, ringworm, melisma, impetigo, acneaestivalis (Mallorca acne), acne conglobate, acne cosmetica (cosmeticacne), acne fulminans (acute febrile ulcerative acne), acne keloidalisnuchae (acne keloidalis, dermatitis papillaris capillitii, folliculitiskeloidalis, folliculitis keloidis nuchae, nuchal keloid acne), adultforehead with scattered red pimples, acne vulgaris, acne mechanica, acnemedicamentosa, acne miliaris necrotica (acne varioliformis), acnevulgaris, acne with facial edema (solid facial edema), blepharophyma,erythrotelangiectatic rosacea (erythematotelangiectatic rosacea,vascular rosacea), excoriated acne (acne excoriee des jeunes filles,Picker's acne), glandular rosacea, gnathophyma, gram-negative rosacea,granulomatous facial dermatitis, rhinophyma, granulomatous perioraldermatitis, halogen acne, hidradenitis suppurativa (acne inversa,pyoderma fistulans significa, Verneuil's disease), idiopathic facialaseptic granuloma, infantile acne, lupoid rosacea (granulomatousrosacea, micropapular tuberculid, rosacea-like tuberculid ofLewandowsky), lupus miliaris disseminatus faciei, metophyma, neonatalacne (acne infantum, acne neonatorum, neonatal cephalic pustulosis),occupational acne, oil acne, ocular rosacea (ophthalmic rosacea,ophthalmorosacea), otophyma, periorificial dermatitis, persistent edemaof rosacea (chronic upper facial erythematous edema, Morbihan's disease,rosaceous lymphedema), phymatous rosacea, pomade acne, papulopustularrosacea (inflammatory rosacea), perifolliculitis capitis abscedens etsuffodiens (dissecting cellulitis of the scalp, dissecting folliculitis,perifolliculitis capitis abscedens et suffodiens of Hoffman), perioraldermatitis, periorbital dermatitis (periocular dermatitis), pyodermafaciale (rosacea fulminans), rhinophyma, rosacea (acne rosacea), rosaceaconglobate, synovitis-acne-pustulosis-hyperostosis-osteomyelitissyndrome (SAPHO syndrome), steroid rosacea, tar acne, skin cancer,tropical acne or a combination thereof. In some embodiments, the subjecthas wrinkles, acne scars, reduced skin elasticity, sagging skin,increased skin dryness, rashes, redness, translucency, fine lines, lossof radiance, increase in the dullness of skin, uneven pigmentation,discoloration, blotchiness, scarring, rough and leathery appearance,freckles, moles, actinic keratosis, slower wound healing, easy bruisingand tearing, ruddiness, uneven texture, fine lines, age spots, or acombination thereof.

In some embodiments, the base composition is a topical cosmetic base isa serum, lotion, liquid primer, cream, gel or a combination thereof. Insome embodiments, the base composition further includes at least onekeratolytic agent, at least one anti-inflammatory agent, sun protectionagent, preservative for platelets in the platelet rich plasma, nutrientor a combination thereof.

In some embodiments, the activating step further includes degranulatingplatelets in the platelet rich plasma of the first topical formulationand releasing growth factors from the platelets into and through theskin of the subject. The degranulation may lead to release of threemajor types of secretory granules in platelets, such as lysosomes, alphaand dense granules.

Application of the second formulation may be applied 5, 10, 20, 30, 40,50, 60, 70, 80, 90 or 120 days after application of the firstformulation. In some embodiments, the second formulation may be appliedevery five days after the first application of the first formulation forup to 120 days. In some embodiments, the first formulation is reappliedto an area of skin that is afflicted with the skin disorder or to anarea of skin that is showing signs of aging, and the second formulationis applied over the area 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 120days after application of the first formulation. The second formulationmay not be applied to the ears, eyes, mouth area or other body orifices.In some embodiments, wherein the second formulation includes thrombin,the second formulation may not be applied to the ears, eyes, mouth areaor other body orifices.

In some embodiments, a method of treating skin on a subject sufferingfrom skin damage and/or signs of aging is provided. The method comprisesobtaining whole blood from a subject having skin damage, centrifugingthe blood to separate platelet rich plasma from platelet poor plasma andred blood cells, collecting the platelet rich plasma (PRP), adding atopical cosmetic base, gel, serum, and cream or combination thereof tothe platelet rich plasma to form a solution thereby forming a firsttopical formulation, providing the first topical formulation to thesubject for treatment of the skin damage for a 90 day time periodwherein the providing includes instructions for the subject to topicallyapply the first topical formulation to an area of the skin and skindamage multiple times a day, activating the platelet rich plasma,wherein the activating includes providing a second topical formulationto the subject or changing the temperature of the base compositionthereby activating the PRP through a temperature change; wherein theproviding includes applying the second topical formulation over the areaof the skin and skin damage that has received the first topicalformulation, wherein treating the skin damage and signs of aging isselected from the group consisting of diminishing pore size, diminishingbacteria damage, diminishing red areas, reducing skin damage caused bysun, and maintaining or improving skin tone. The method furthercomprises providing topical formulation to the subject comprising ofcalcium, sodium, or other platelet rich plasma activator to be appliedonto base and platelet rich plasma admixed solution. In someembodiments, the first formulation is applied 1, 2, 3 or 4 times a day.In some embodiments, the first formulation is applied for up to 30, 60,90 or 120 days or any number of days in between a range defined by anytwo aforementioned values. In some embodiments, the first formulation isapplied for up to 90, 120, 150, 200 or 250 days. The first and secondformulation may not be applied to the ears, eyes, mouth area or otherorifices. In some embodiments, wherein the second formulation includesthrombin, the second formulation may not be applied to the ears, eyes,mouth area or other orifices.

In some embodiments, the second formulation is a gel, liquid, cream orlotion. The second formulation may be applied over the area of skin thathas received the first formulation in order to activate the platelets inthe first formulation.

In some embodiments, the second formulation is a liquid and wherein theliquid is applied as a spray such as an aerosol spray or pump spray. Insome embodiments, the second formulation includes a platelet rich plasmaactivator. In some embodiments, the second formulation includes calcium,magnesium, sodium, components of snake venom or combinations thereof. Insome embodiments, the component of snake venom comprises Batroxobin. Insome embodiments, the second formulation further includes Batroxobin,epinephrine and/or thrombin. The calcium may be provided as calciumchloride or calcium carbonate, for example. In some embodiments, thesecond formulation comprises 10% calcium (e.g. calcium ion, calciumchloride calcium carbonate, calcium gluconate). In some embodiments, thesecond formulation comprises 15%-20% ethanol, 1:4 ratio autologousthrombin to PRP, 10% calcium chloride w/ 10,000 units of bovinethrombin, batroxobin, chitosan, or any combination thereof. In someembodiments, wherein the second formulation is provided as a spray, thesecond formulation comprises Aqua (Water), a platelet activator,Polysorbate 80, Propylene Glycol, Sodium Chloride, Anthemis nobilis(Chamomile) Flower Oil, Rosa canina (Rose Hip) Fruit Oil, Thymusvulgaris (Thyme) Flower/Leaf Oil, Althaea officinalis (Marshmallow) RootExtract, Hamamelis virginiana (Witch Hazel) Water, Alcohol, Glycerin,Ethylhexylglycerin, Citric Acid, Potassium Sorbate, Phenoxyethanol. Insome embodiments, the platelet activator is calcium chloride. The secondformulation may not be applied to the ears, eyes, mouth area or otherbody orifices. In some embodiments, wherein the second formulationcomprises thrombin, the second formulation may not be applied to theears, eyes, mouth area or other body orifices.

In some embodiments, the subject is also suffering from a skin disorder.In some embodiments, the skin damage and signs of aging are caused bythe skin disorder. In some embodiments, the skin damage and signs ofaging are caused by smoking, alcohol, diet, extreme temperatures,chemicals, stress, lack of sleep, poor diet, poor immune system or acombination thereof.

In some embodiments, the skin disorder is selected from a groupconsisting of stretch marks (striae), psoriasis, skin cancer, acne,alopecia, carbuncles, dermatitis, eczema, atopic dermatitis, contactdermatitis, seborrheic dermatitis, cradle cap, perioral dermatitis,shingles, ringworm, melisma, impetigo, acne aestivalis (Mallorca acne),acne conglobate, acne cosmetica (cosmetic acne), acne fulminans (acutefebrile ulcerative acne), acne keloidalis nuchae (acne keloidalis,dermatitis papillaris capillitii, folliculitis keloidalis, folliculitiskeloidis nuchae, nuchal keloid acne), adult forehead with scattered redpimples, acne vulgaris, acne mechanica, acne medicamentosa, acnemiliaris necrotica (acne varioliformis), acne vulgaris, acne with facialedema (solid facial edema), blepharophyma, erythrotelangiectatic rosacea(erythematotelangiectatic rosacea, vascular rosacea), excoriated acne(acne excoriee des jeunes filles, Picker's acne), glandular rosacea,gnathophyma, gram-negative rosacea, granulomatous facial dermatitis,rhinophyma, granulomatous perioral dermatitis, halogen acne,hidradenitis suppurativa (acne inversa, pyoderma fistulans significa,Verneuil's disease), idiopathic facial aseptic granuloma, infantileacne, lupoid rosacea (granulomatous rosacea, micropapular tuberculid,rosacea-like tuberculid of Lewandowsky), lupus miliaris disseminatusfaciei, metophyma, neonatal acne (acne infantum, acne neonatorum,neonatal cephalic pustulosis), occupational acne, oil acne, ocularrosacea (ophthalmic rosacea, ophthalmorosacea), otophyma, periorificialdermatitis, persistent edema of rosacea (chronic upper facialerythematous edema, Morbihan's disease, rosaceous lymphedema), phymatousrosacea, pomade acne, papulopustular rosacea (inflammatory rosacea),perifolliculitis capitis abscedens et suffodiens (dissecting cellulitisof the scalp, dissecting folliculitis, perifolliculitis capitisabscedens et suffodiens of Hoffman), perioral dermatitis, periorbitaldermatitis (periocular dermatitis), pyoderma faciale (rosaceafulminans), rhinophyma, rosacea (acne rosacea), rosacea conglobate,synovitis-acne-pustulosis-hyperostosis-osteomyelitis syndrome (SAPHOsyndrome), steroid rosacea, tar acne, skin cancer, tropical acne or acombination thereof.

In some embodiments, the subject has stretch marks, wrinkles, acnescars, reduced skin elasticity, sagging skin, increased skin dryness,rashes, redness, translucency, fine lines, loss of radiance, increase inthe dullness of skin, uneven pigmentation, discoloration, blotchiness,scarring, rough and leathery appearance, freckles, moles, actinickeratosis, slower wound healing, easy bruising and tearing, ruddiness,uneven texture, fine lines, age spots, or a combination thereof.

In some embodiments, the base composition is a serum, lotion, liquidprimer, cream, gel or a combination thereof.

In some embodiments, the base composition further comprises at least onekeratolytic agent, at least one anti-inflammatory agent, sun protectionagent, preservative for platelets in the platelet rich plasma, nutrientor a combination thereof.

In some embodiments, the activating step further comprises degranulatingplatelets in the platelet rich plasma of the first topical formulationand releasing growth factors from the platelets into and through theskin of the subject.

In some embodiments, the second formulation comprises a platelet richplasma activator. In some embodiments, the second formulation is aliquid, and wherein the liquid is in a spray bottle. In someembodiments, the second formulation comprises calcium, magnesium,sodium, components of snake venom or combinations thereof. In someembodiments, the component of snake venom comprises Batroxobin. Thecalcium may be provided as calcium chloride or calcium carbonate, forexample. In some embodiments, the kit stored at 4° C. to preserve thefirst and second formulation. In some embodiments, wherein the secondformulation does not include snake venom, the kit does not need to bestored in refrigeration. In some embodiments, the second formulationcomprises 10% calcium (e.g. calcium ion, calcium chloride calciumcarbonate, calcium gluconate). In some embodiments, the secondformulation comprises 15%-20% ethanol, 1:4 ratio autologous thrombin toPRP, 10% calcium chloride w/ 10,000 units of bovine thrombin,batroxobin, chitosan, or any combination thereof. The second formulationmay not be applied to the ears, eyes, mouth area or other orifices. Insome embodiments, wherein the second formulation comprises thrombin, thesecond formulation may not be applied to the ears, eyes, mouth area orother orifices.

Application of the second formulation may be applied 5, 10, 20, 30, 40,50, 60, 70, 80, 90 or 120 days after application of the firstformulation. In some embodiments, the second formulation may be appliedevery five days after the first application of the first formulation forup to 120 days. In some embodiments, the first formulation is reappliedto an area of skin that is afflicted with the skin disorder or to anarea of skin that is showing signs of aging, and the second formulationis applied over the area 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 120days after application of the first formulation. The second formulationmay not be applied to the ears, eyes, mouth area or other orifices. Insome embodiments, wherein the second formulation comprises thrombin, thesecond formulation may not be applied to the ears, eyes, mouth area orother orifices.

In some embodiments, the method of treating skin further includessubjecting the skin to skin obstruction, such as electroporation,radiofrequency, ultrasound, high intensity focused ultrasound (HIFU),intense pulsed light (IPL), ablative laser, non-ablative laser,microdermabrasion, hydradermabrasion, iontophoresis, chemical peel,plasma, high velocity air, high velocity aqueous solution, or needling,or a combination thereof. In some embodiments, skin obstruction takesplace before administration of the first formulation, duringadministration of the first formulation, after administration of thefirst formulation but prior to administration of the second formulation,during administration of the second formulation, or after administrationof the second formulation. In some embodiments, skin obstruction takesplace for a period of time of about 0.5 minutes to 20 minutes, such as0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 minutes, or for an amount of time within a range defined by anytwo of the aforementioned values. In some embodiments, skin obstructiontakes place using a therapeutic skin obstruction device that may be usedon skin of a subject. In some embodiments, skin obstruction prior to,concomitantly with, or following application of the formulationsgenerates channels through which the biologic, such as the PRP canpenetrate into the viable epidermis and dermis to improve a skincondition.

In any of the methods provided herein, the method may further includeproviding a low concentration acidic spray to be applied to thecomposition following topical application of the composition to thesubject. In some embodiments, the low concentration acid spray includeslow concentrations of hydrochloric acid (HCl), such as hydrochloric acidin an amount ranging from about 0.01% to about 10% HCl, such as 0.01%,0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or in anamount with a range defined by any two of the aforemented values.

Formulations Comprising Fibroblasts

In some embodiments, a method of treating a subject suffering from skindamage and signs of aging is provided. The method comprise obtainingfibroblast skin cells from skin of a subject, placing the fibroblastskin cells in growth media, growing the fibroblast skin cells up toconfluency, mixing the fibroblast cells with a base composition whereinthe base composition preserves, extends the life of, enhances,maintains, sustains, or otherwise prolongs the cells, thereby making afirst formulation comprising fibroblast cells, and applying theformulation to the skin of the subject. In some embodiments, the basecomposition preserves, extends the life of, enhances, maintains,sustains, or otherwise prolongs the cells for a period of more than 30,40, 50, 60, 70, 80, 90, 100, or 120 days.

In some embodiments, the subject is also suffering from a skin disorder.In some embodiments, the skin damage and signs of aging are caused bythe skin disorder. In some embodiments, the skin damage and signs ofaging are caused by smoking, alcohol, diet, extreme temperatures,chemicals, stress, lack of sleep, poor diet, poor immune system or acombination thereof. In some embodiments, the skin disorder is selectedfrom a group consisting of stretch marks (striae), psoriasis, skincancer, acne, alopecia, carbuncles, dermatitis, eczema, atopicdermatitis, contact dermatitis, seborrheic dermatitis, cradle cap,perioral dermatitis, shingles, ringworm, melisma, impetigo, acneaestivalis (Mallorca acne), acne conglobate, acne cosmetica (cosmeticacne), acne fulminans (acute febrile ulcerative acne), acne keloidalisnuchae (acne keloidalis, dermatitis papillaris capillitii, folliculitiskeloidalis, folliculitis keloidis nuchae, nuchal keloid acne), adultforehead with scattered red pimples, acne vulgaris, acne mechanica, acnemedicamentosa, acne miliaris necrotica (acne varioliformis), acnevulgaris, acne with facial edema (solid facial edema), blepharophyma,erythrotelangiectatic rosacea (erythematotelangiectatic rosacea,vascular rosacea), excoriated acne (acne excoriee des jeunes filles,Picker's acne), glandular rosacea, gnathophyma, gram-negative rosacea,granulomatous facial dermatitis, rhinophyma, granulomatous perioraldermatitis, halogen acne, hidradenitis suppurativa (acne inversa,pyoderma fistulans significa, Verneuil's disease), idiopathic facialaseptic granuloma, infantile acne, lupoid rosacea (granulomatousrosacea, micropapular tuberculid, rosacea-like tuberculid ofLewandowsky), lupus miliaris disseminatus faciei, metophyma, neonatalacne (acne infantum, acne neonatorum, neonatal cephalic pustulosis),occupational acne, oil acne, ocular rosacea (ophthalmic rosacea,ophthalmorosacea), otophyma, periorificial dermatitis, persistent edemaof rosacea (chronic upper facial erythematous edema, Morbihan's disease,rosaceous lymphedema), phymatous rosacea, pomade acne, papulopustularrosacea (inflammatory rosacea), perifolliculitis capitis abscedens etsuffodiens (dissecting cellulitis of the scalp, dissecting folliculitis,perifolliculitis capitis abscedens et suffodiens of Hoffman), perioraldermatitis, periorbital dermatitis (periocular dermatitis), pyodermafaciale (rosacea fulminans), rhinophyma, rosacea (acne rosacea), rosaceaconglobate, synovitis-acne-pustulosis-hyperostosis-osteomyelitissyndrome (SAPHO syndrome), steroid rosacea, tar acne, skin cancer,tropical acne or a combination thereof. In some embodiments, the subjecthas wrinkles, acne scars, reduced skin elasticity, sagging skin,increased skin dryness, rashes, redness, translucency, fine lines, lossof radiance, increase in the dullness of skin, uneven pigmentation,discoloration, blotchiness, scarring, rough and leathery appearance,freckles, moles, actinic keratosis, slower wound healing, easy bruisingand tearing, ruddiness, uneven texture, fine lines, age spots, or acombination thereof.

In some embodiments, the base composition is a serum, lotion, liquidprimer, cream, gel, or liquid or a combination thereof. In someembodiments, the base composition further comprises at least onekeratolytic agent, at least one anti-inflammatory agent, sun protectionagent, or a combination thereof.

In some embodiments, the fibroblast cells are obtained from the skin onthe neck, arms, legs, buttocks, stomach, back or behind the ear of thesubject. In some embodiments, the method further comprises freezing thefibroblast cells and storing the fibroblast cells prior to mixingfibroblast cells with the topical cosmetic base.

In some embodiments, the method further comprising thawing thefibroblast cells prior to mixing the fibroblast cells with the basecomposition.

In some embodiments, the growth media comprises human platelet lysate,platelet rich plasma, human serum or a combination thereof.

In some embodiments, growth media comprises human platelet lysate andwherein the human platelet lysate is from the subject, wherein the humanplatelet lysate is obtained by: obtaining blood from the subject,centrifuging the blood to separate platelet-poor plasma, platelet richplasma; and collecting the platelet rich plasma.

In some embodiments, the method further comprises subjecting theplatelet rich plasma to freeze thaw cycles, thereby forming humanplatelet lysate.

In some embodiments, the method further comprises adding mixing theplatelet rich plasma, human platelet lysate or a combination thereof,with growth media, prior to placing the fibroblast skin cells in thegrowth media.

In some embodiments, the method further comprises activating plateletsin the platelet rich plasma. The method comprises providing a secondformulation to the subject, wherein the providing comprises applying thesecond topical formulation over the area of the skin and skin damagethat has received the first topical formulation.

In some embodiments, activating platelets in the PRP includes changingthe temperature of the first topical formulation from a refrigeratedtemperature to room temperature, thereby activating the plateletsthrough a change in temperature. In some embodiments, changing thetemperature of the first topical formulation includes application of theformulation to a subject, wherein the application increases thetemperature of the formulation, thereby activating the platelets.

In some embodiments, the activating comprises degranulating platelets inthe platelet rich plasma of the first topical formulation and releasinggrowth factors from the platelets into the skin of the subject.

In some embodiments, the second formulation is a gel, liquid, cream orlotion. In some embodiments, the second formulation is a liquid andwherein the liquid is applied as a spray such as an aerosol spray orpump spray. In some embodiments, the second formulation comprises aplatelet rich plasma activator. In some embodiments, the platelet richplasma activator calcium, magnesium, sodium, components of snake venomor combinations thereof. In some embodiments, the component of snakevenom comprises Batroxobin. The calcium may be provided as calciumchloride or calcium carbonate, for example. In some embodiments, thesecond formulation comprises calcium, magnesium, sodium, components ofsnake venom or combinations thereof. In some embodiments, the snakevenom comprises oxydoreductases, transferases, hydrolases, or lyases ora combination thereof. In some embodiments, the snake venom comprisesdehydrogenase lactate, L-amino-acid oxidase, Catalase, Alanine aminotransferase, Phospholipase A2, Lysophospholipase, Acetylcholinesterase,Alkaline phosphatase, Acid phosphatase, 5′-Nucleotidase,Phosphodiesterase, Deoxyribonuclease, Ribonuclease 1, Adenosinetriphosphatase, Amylase, Hyaluronidase, NAD-Nucleotidase, Kininogenase,Factor-X activator, Heparinase, α-Fibrinogenase, β-Fibrinogenase,Fibrinolytic enzyme, Prothrombin activator, Collagenase, Elastase orGlucosamine ammonium lyase or combinations thereof. In some embodiments,the snake venom comprises α-neurotoxins, β-neurotoxins, κ-Toxins,Dendrotoxins, Cardiotoxins, Myotoxins, Sarafotoxins or Hemorrhagins, orcombinations thereof. In some embodiments the snake venom comprisesα-Bungarotoxin, α-toxin, erabutoxin, cobratoxin, Notexin, ammodytoxin,β-Bungarotoxin, crotoxin, taipoxin, κ-Toxin, Dendrotoxin, toxins I andK, y-Toxin, cardiotoxin, cytotoxin, Myotoxin-a, crotamine, Sarafotoxina, Sarafotoxin b, Sarafotoxin c, Phospholipase A2, mucrotoxin A,hemorrhagic toxins, HT1 or HT2 or combinations thereof. In someembodiments, the second formulation further comprises Batroxobin,epinephrine and/or thrombin. In some embodiments, the snake venomcomprises Batroxobin. In some embodiments, the second formulationcomprises 10% calcium (e.g. calcium ion, calcium chloride calciumcarbonate, calcium gluconate). In some embodiments, the secondformulation comprises 15%-20% ethanol, 1:4 ratio autologous thrombin toPRP, 10% calcium chloride w/ 10,000 units of bovine thrombin,batroxobin, chitosan, or any combination thereof. In some embodiments,wherein the second formulation is provided as a spray, the secondformulation comprises Aqua (Water), a platelet activator, Polysorbate80, Propylene Glycol, Sodium Chloride, Anthemis nobilis (Chamomile)Flower Oil, Rosa canina (Rose Hip) Fruit Oil, Thymus vulgaris (Thyme)Flower/Leaf Oil, Althaea officinalis (Marshmallow) Root Extract,Hamamelis virginiana (Witch Hazel) Water, Alcohol, Glycerin,Ethylhexylglycerin, Citric Acid, Potassium Sorbate, Phenoxyethanol. Insome embodiments, the platelet activator is calcium chloride.

In some embodiments, the subject is also suffering from a skin disorder.In some embodiments, the skin damage and signs of aging are caused bythe skin disorder. In some embodiments, the skin damage and signs ofaging are caused by smoking, alcohol, diet, extreme temperatures,chemicals, stress, lack of sleep, poor diet, poor immune system or acombination thereof. In some embodiments, the skin disorder is selectedfrom a group consisting of stretch marks (striae), psoriasis, skincancer, acne, alopecia, carbuncles, dermatitis, eczema, atopicdermatitis, contact dermatitis, seborrheic dermatitis, cradle cap,perioral dermatitis, shingles, ringworm, melisma, impetigo, acneaestivalis (Mallorca acne), acne conglobate, acne cosmetica (cosmeticacne), acne fulminans (acute febrile ulcerative acne), acne keloidalisnuchae (acne keloidalis, dermatitis papillaris capillitii, folliculitiskeloidalis, folliculitis keloidis nuchae, nuchal keloid acne), adultforehead with scattered red pimples, acne vulgaris, acne mechanica, acnemedicamentosa, acne miliaris necrotica (acne varioliformis), acnevulgaris, acne with facial edema (solid facial edema), blepharophyma,erythrotelangiectatic rosacea (erythematotelangiectatic rosacea,vascular rosacea), excoriated acne (acne excoriee des jeunes filles,Picker's acne), glandular rosacea, gnathophyma, gram-negative rosacea,granulomatous facial dermatitis, rhinophyma, granulomatous perioraldermatitis, halogen acne, hidradenitis suppurativa (acne inversa,pyoderma fistulans significa, Verneuil's disease), idiopathic facialaseptic granuloma, infantile acne, lupoid rosacea (granulomatousrosacea, micropapular tuberculid, rosacea-like tuberculid ofLewandowsky), lupus miliaris disseminatus faciei, metophyma, neonatalacne (acne infantum, acne neonatorum, neonatal cephalic pustulosis),occupational acne, oil acne, ocular rosacea (ophthalmic rosacea,ophthalmorosacea), otophyma, periorificial dermatitis, persistent edemaof rosacea (chronic upper facial erythematous edema, Morbihan's disease,rosaceous lymphedema), phymatous rosacea, pomade acne, papulopustularrosacea (inflammatory rosacea), perifolliculitis capitis abscedens etsuffodiens (dissecting cellulitis of the scalp, dissecting folliculitis,perifolliculitis capitis abscedens et suffodiens of Hoffman), perioraldermatitis, periorbital dermatitis (periocular dermatitis), pyodermafaciale (rosacea fulminans), rhinophyma, rosacea (acne rosacea), rosaceaconglobate, synovitis-acne-pustulosis-hyperostosis-osteomyelitissyndrome (SAPHO syndrome), steroid rosacea, tar acne, skin cancer,tropical acne or a combination thereof. In some embodiments, the subjecthas wrinkles, acne scars, reduced skin elasticity, sagging skin,increased skin dryness, rashes, redness, translucency, fine lines, lossof radiance, increase in the dullness of skin, uneven pigmentation,discoloration, blotchiness, scarring, rough and leathery appearance,freckles, moles, actinic keratosis, slower wound healing, easy bruisingand tearing, ruddiness, uneven texture, fine lines, age spots, or acombination thereof.

In some embodiments, the topical cosmetic base is a serum, lotion,liquid primer, cream, gel or a combination thereof. In some embodiments,the topical cosmetic base further comprises at least one keratolyticagent, at least one anti-inflammatory agent, sun protection agent,preservative for platelets in the platelet rich plasma, nutrient or acombination thereof.

In some embodiments, the activating step further comprises degranulatingplatelets in the platelet rich plasma of the first topical formulationand releasing growth factors from the platelets into and through theskin of the subject. The degranulation may lead to release of threemajor types of secretory granules in platelets, such as lysosomes, alphaand dense granules.

Application of the second formulation may be applied 5, 10, 20, 30, 40,50, 60, 70, 80, 90 or 120 days after application of the firstformulation. In some embodiments, the second formulation may be appliedevery five days after the first application of the first formulation forup to 120 days. In some embodiments, the first formulation is reappliedto an area of skin that is afflicted with the skin disorder or to anarea of skin that is showing signs of aging, and the second formulationis applied over the area 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 120days after application of the first formulation.

Advantages of Using the Inactivated First Formulation with theActivation Spray

An advantage of the first formulation is that the first formulation isnot activated when first applied, which allows the absorption of thetopical formulation into the skin and to the area with skin damage. Thesecond formulation to activate the first formulation is in a liquid. Theliquid may be administered as a spray, such as an aerosol spray. Afterwhich the spray is applied to activate or degranulate the platelets inthe first formulation. The advantage of the spray would be to preventtouching or irritating skin damage, as the formulation applied directlyto the affected area could cause additional irritation from rubbing aswell as contamination and infection of the skin site. Thus the firstformulation is absorbed first without being bound to any theory, thisfirst absorption allows the absorption of the growth factors to bereleased into and through the skin to enhance the healing of a skindisorder of for preventing signs of aging in skin after the spraycomprising the activator is applied. In some embodiments, the firstformulation is allowed to absorb prior to applying the secondformulation. The first formulation may be applied 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hoursprior to adding the second formulation. In some embodiments, thewaterproof dressing is applied onto the skin after the addition of thefirst formulation to prevent contamination or to prevent wash off.Depending on the type of damage of the skin, the first formulation isallowed to be absorbed for some time in order to allow penetration ofthe growth factors that are released during degranulation. For example,if the damage is extended deep into the epidermis or dermis, the firstformulation may be allowed to absorb on the skin for at least 24 hoursprior to allowing degranulation by applying the second formulation. Insome embodiments, the second formulation is applied on top of the firstformulation right away to start the activation process of the plateletsby the platelet activator. In some embodiments, right away is within afew seconds to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes after applicationof the first formulation. In some embodiments, the first formulation maybe applied 1, 2, 3, 4, 5, 6, days or even 1 or 2 weeks prior toadministration of the second formulation.

The use of an inactivated first formulation increases the lifespan ofthe platelets within the PRP and therefore the lifespan of the plateletfactors essential for skin rejuvenation because the platelet factorsstay dormant within the granules of the cell. The lifespan of theplatelet factors is prolonged due to the components of the basecomposition, which is capable of preserving, extending the life of,enhancing, maintaining, sustaining, or otherwise prolonging theplatelets and platelet factors for a period of more than 30, 40, 50, 60,70, 80, 90, 100, or 120 days. Therefore, activating the firstformulation with the second formulation increases the efficacy of firstformulation when the platelets of the first formulation remaininactivated.

In some embodiments, the first formulation may also be applied as aspray, to reduce contact with skin damage and to protect againstadditional irritation from rubbing as well as contamination andinfection of the skin site.

Activation of the PRP after the first formulation is on the skin wouldallow one to regulate the growth factors release and endothelial celldivision. During activation or degranulation, platelet derived growthfactor, vascular endothelial growth factor (VEGF), transforming growthfactor beta (TGF-beta), and interleukin 1 beta levels are released.

In some embodiments, the compositions provided herein preserve, extendthe life of, enhance, maintain, sustain, or otherwise prolong thebiologic, such as cells, proteins, nucleic acids, growth factors,antibodies, blood, serum, plasma, or components thereof, or otherbiomolecules. In some embodiments, biomolecules include PDGF, VEG, IGF,TGF-B1, FGF, EGF, CTGF PDGF-A, PDGF-AB, PDGF-B, CTGF, CTAP-3, basicfibroblast GF, TGF-B1, PF4, PDAF, endothelial cell growth inhibitor,EPF, EGI, KGF, ANGPTL6, IGF, IGFBP-3, TGF-B2, Estrogen receptor-relatedprotein, VEGF, f-ECGF, HGF, Histamine-releasing factors, Humancollagenase inhibitor, fibronectin, PMP-1, t-PMP, TC1, TC2, Vitronectin,Thrombospondin, Serotonin, Cathepsin, CXCL7, NAP-2, NAP-4, SST, RANTES,CTAP-3, PP14, SCUBE1, CCN family, Annexin 11, HSP27, HSP60, vonWillebrand factor, Albumin, Immunoglobulins IgG, IgM, IgA, Coagulationfactors V, VII, XI, XIII, Fibrinogen, Histamine, ATP, ADP, GPT, GDP,Collagenase, ADAMTS-13. Superoxide dismutase (SOD), Heparinase, α1-α2anti/trypsin, α2-antiplasmin, α2-macroglobulin, C1-INH, aldolase,carboxypeptidases, acid phosphatase, arylsulphatase, β-galactoidase,β-glucoronidase, β-glycerolphosphatase, α/β-glucosidases,α/β-fucosidases, α-mannosidase, α-arabinosidase, SIG, JE, KC, inducibleBMP-2, -6, -7, Metalloprotease MMP-1, -2, -9, -13, ECM remodelingfactors, ERK, PC, LPA, or HMGB1 or a combination thereof. Componentsreleased after degranulation is provided in Borzini et al (Borzini, P.and Mazzucco, I., 2007. Platelet-rich plasma (PRP) and plateletderivatives for topical therapy. What is true from the biologic viewpoint? ISBT Science Series, 2(1), pp. 272-281; incorporated by referencein its entirety herein.

Advantages of administering the second formulation as a spray also leadsto a better coverage of the first layer comprising the first formulationon the skin, thus allowing the all the cells of the first formulation tobe degranulated. Providing the second formulation as a spray preventsdisturbing the first formulation layer and irritating the cells of thefirst formulation that is already in the skin. Additionally, a spray maylead to even coverage over the skin allowing an improved efficacy of thetwo formulations to work together.

First Formulation

In some embodiments, the first formulation comprises a base compositionthat comprises a humectant, sodium bicarbonate, a buffering agent,magnesium chloride, and a viscosity modifying agent. In someembodiments, the first formulation further includes a skin conditioningagent, an emulsion stabilizer, a chelating agent, a biocide, a biologicbuffering agent, an antioxidant, a pH adjuster, a sun protection agent,a cell culture reagent, an enzyme inhibitor, an activation inhibitor, askin permeability enhancer, a skin delivery system, or a combinationthereof. In some embodiments, the base composition comprises aqua(water), Lonicera japonica (honeysuckle) flower extract, glycerin,niacinamide, polyacrylate crosspolymer-6, Lonicera caprifolium(honeysuckle) flower extract, Persea gratissima (avocado) oil, Aloebarbadensis leaf juice, sodium gluconate, sodium hyaluronate (L),Althaea officinalis (marshmallow) root extract, O-cymen-5-OL, sodiumhydroxide, glucose (D), sodium chloride, sodium citrate, sodium acetate,sodium bicarbonate, tocopheryl acetate (D-alpha), potassium phosphate,potassium chloride, magnesium chloride, phenoxyethanol, Anthemis nobilis(chamomile) flower oil, Rosmarinus officinalis (rosemary) leaf oil,Thymus vulgaris (thyme) flower/leaf oil, benzyl alcohol, caprylylglycol, caprylhydroxamic acid, or combinations thereof. In someembodiments, any of the aforementioned components are present in anamount from about 0.001 wt % to about 99% wt %, including 0.001, 0.002,0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4,0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 96, 97, 98, or 99%, or an amount within a rangedefined by any two of the aforementioned values. In some embodiments,any of the aforementioned components are present in a composition in atherapeutically effective amount. For example, any of the aforementionedcomponents may be present in the base composition in an amount fromabout 0.001 mg to about 10 mg, such as 0.001, 0.05, 0.1, 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 mg, or an amount within a range defined by anytwo of the aforementioned values.

In some embodiments, the first formulation is provided to a subject inneed in an amount of about 0.05 mg to about 10 mg, such as 0.05, 0.06,0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 mg, or an amount within a range defined by anytwo of the aforementioned values. One of skill in the art will recognizethat the amount that is provided to a subject will depend on thecondition being treated, the severity of the condition, the size or areaof the region being treated, or other such considerations, and thus, theamount or quantity of first formulation that is applied to a subject mayvary.

Importantly, the base composition is capable of preserving, extendingthe life of, enhancing, maintaining, sustaining, or otherwise prolonginga biologics with which it is mixed to form the first formulation, suchthat the biologic has an extended life or activity when mixed with thebase composition. In some embodiments, the biologic has an extended lifeor activity for a period of more than 30, 40, 50, 60, 70, 80, 90, 100,or 120 days, such that the first formulation may be applied oradministered to a subject in need many days, weeks, or months afterpreparation of the formulation. This is in contrast to currentlyavailable compositions, which have a short shelf-life due to theinability of the biologic to maintain viability or activity over time.The base composition described herein enables increased longevity andactivity due to the specific components and quantities of componentsprovided and described herein.

Following application of a first formulation to a subject in needthereof, the biologic, such as PRP, may be activated by application ofthe second formulation.

Second Formulation

In some embodiments, the second formulation is provided as a spray, thesecond formulation comprises aqua (Water), a platelet activator,Polysorbate 80, Propylene Glycol, Sodium Chloride, Anthemis nobilis(Chamomile) Flower Oil, Rosa canina (Rose Hip) Fruit Oil, Thymusvulgaris (Thyme) Flower/Leaf Oil, Althaea officinalis (Marshmallow) RootExtract, Hamamelis virginiana (Witch Hazel) Water, Alcohol, Glycerin,Ethylhexylglycerin, Citric Acid, Potassium Sorbate, Phenoxyethanol. Insome embodiments, the platelet activator is calcium chloride.

In some embodiments, the first and/or second formulation comprises skinpenetrators, preservatives and/or botanicals. In some embodiments, theskin penetrators include Fulvate fractions (Omni BioceuticalInnovations) liposomes, nanoparticles, dimethylsulfoxide, sodium laurylsulfate, hyaluronic acid, nanostructured lipid.

In some embodiments of the first or second formulation, the formulationmay be provided within a spray or aerosol bottle. Formulation 1 maycomprise aqua (water), Lonicera japonica (honeysuckle) flower extract,glycerin, niacinamide, polyacrylate crosspolymer-6, Lonicera caprifolium(honeysuckle) flower extract, Persea gratissima (avocado) oil, Aloebarbadensis leaf juice, sodium gluconate, sodium hyaluronate (L),Althaea officinalis (marshmallow) root extract, O-cymen-5-OL, sodiumhydroxide, glucose (D), sodium chloride, sodium citrate, sodium acetate,sodium bicarbonate, tocopheryl acetate (D-alpha), potassium phosphate,potassium chloride, magnesium chloride, phenoxyethanol, Anthemis nobilis(chamomile) flower oil, Rosmarinus officinalis (rosemary) leaf oil,Thymus vulgaris (thyme) flower/leaf oil, benzyl alcohol, caprylylglycol, caprylhydroxamic acid, or combinations thereof. In someembodiments, any of the aforementioned components are present in anamount from about 0.001 wt % to about 99% wt %, including 0.001, 0.002,0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4,0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 96, 97, 98, or 99%, or an amount within a rangedefined by any two of the aforementioned values.

Platelet buffering ingredients may include magnesium chloride, potassiumchloride, glucose, sodium bicarbonate, sodium chloride, sodium citrate,sodium acetate, sodium phosphate, Fetal Bovine Serum (FBS). These canalso be included for the first formulation.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure. Those in the art willappreciate that many other embodiments also fall within the scope of thedisclosure, as it is described herein above and in the claims.

Example 1: Preparation of a Base Composition

The following example demonstrates preparation of a base compositionthat preserves, extends the life of, enhances, maintains, sustains, orotherwise prolongs biologics for extended periods of time.

The base composition was prepared by mixing the ingredients provided inTable 1.

TABLE 1 Component Wt % Water 92.72 Polyacrylate crosspolymer-6 1.3Lonicera japonica (honeysuckle) flower extract 1.2 Glycerin 1.2Niacinamide 1 Lonicera caprifolium (honeysuckle) flower extract 0.5Persea gratissima (avocado) oil 0.5 Aloe barbadensis leaf juice 0.5Citric acid 0.3 Sodium gluconate 0.2 Sodium hyaluronate (L) 0.1 Althaeaofficinalis (marshmallow) root extract 0.1 O-cymen-5-OL 0.1 Glucose (D)0.1 Sodium chloride 0.01 Sodium citrate 0.01 Sodium acetate 0.01 Sodiumbicarbonate 0.01 Tocopheryl acetate (D-alpha) 0.01 Potassium phosphate0.01 Potassium chloride 0.01 Magnesium chloride 0.01

Example 2: Kits for Formulations for Treating a Skin Disorder or Signsof Aging

In some embodiments, a kit for treating a skin disorder or signs ofaging is provided. The kit comprises a first formulation having atopical cosmetic base, gel, serum, and cream or combination thereof,wherein the first formulation is mixed with platelet rich plasma, andwherein the first formulation further comprises nutrients, botanicals,preservatives or skin penetrators or a combination thereof, and a spraybottle or aerosol container holding a second formulation, the secondformulation including a platelet rich plasma activator in liquidsuspension. In some embodiments of the kit, the kit comprises the secondformulation as a liquid, wherein the liquid is stored in a spraybottled. In some embodiments, the spray is a pump spray or an aerosolspray.

Selection of Subjects for Treatment Formulations

In the embodiments herein, subjects are selected based on their skinailments or disorders which may be selected from stretch marks (striae),psoriasis, skin cancer, acne, alopecia, carbuncles, dermatitis, eczema,atopic dermatitis, contact dermatitis, seborrheic dermatitis, cradlecap, perioral dermatitis, shingles, ringworm, melisma, impetigo, acneaestivalis (Mallorca acne), acne conglobate, acne cosmetica (cosmeticacne), acne fulminans (acute febrile ulcerative acne), acne keloidalisnuchae (acne keloidalis, dermatitis papillaris capillitii, folliculitiskeloidalis, folliculitis keloidis nuchae, nuchal keloid acne), adultforehead with scattered red pimples, acne vulgaris, acne mechanica, acnemedicamentosa, acne miliaris necrotica (acne varioliformis), acnevulgaris, acne with facial edema (solid facial edema), blepharophyma,erythrotelangiectatic rosacea (erythematotelangiectatic rosacea,vascular rosacea), excoriated acne (acne excoriee des jeunes filles,Picker's acne), glandular rosacea, gnathophyma, gram-negative rosacea,granulomatous facial dermatitis, rhinophyma, granulomatous perioraldermatitis, halogen acne, hidradenitis suppurativa (acne inversa,pyoderma fistulans significa, Verneuil's disease), idiopathic facialaseptic granuloma, infantile acne, lupoid rosacea (granulomatousrosacea, micropapular tuberculid, rosacea-like tuberculid ofLewandowsky), lupus miliaris disseminatus faciei, metophyma, neonatalacne (acne infantum, acne neonatorum, neonatal cephalic pustulosis),occupational acne, oil acne, ocular rosacea (ophthalmic rosacea,ophthalmorosacea), otophyma, periorificial dermatitis, persistent edemaof rosacea (chronic upper facial erythematous edema, Morbihan's disease,rosaceous lymphedema), phymatous rosacea, pomade acne, papulopustularrosacea (inflammatory rosacea), perifolliculitis capitis abscedens etsuffodiens (dissecting cellulitis of the scalp, dissecting folliculitis,perifolliculitis capitis abscedens et suffodiens of Hoffman), perioraldermatitis, periorbital dermatitis (periocular dermatitis), pyodermafaciale (rosacea fulminans), rhinophyma, rosacea (acne rosacea), rosaceaconglobate, synovitis-acne-pustulosis-hyperostosis-osteomyelitissyndrome (SAPHO syndrome), steroid rosacea, tar acne, skin cancer,tropical acne or a combination thereof.

Whole blood is obtained from a subject having skin damage which is thencentrifuged to separate platelet rich plasma from platelet poor plasmaand red blood cells. Speeds to separate the PRP from platelet poorplasma can be adjusted accordingly and is a technique known to those ofskill in the art. The platelet rich plasma (PRP) is then collected and abase composition is added such as a topical cosmetic base, a gel, aserum, a lotion, an ointment, a spray, an aerosol, a powder, a solution,a liquid, a foam, a salve, a paste, or a cream or combination thereof toform PRP admixed solution thereby forming a first topical formulation.The base composition preserves, extends, enhances, maintains, sustains,or otherwise prolongs the PRP for a period of more than 30, 40, 50, 60,70, 80, 90, 100, or 120 days, such that the first topical formulationmay be used having functional PRP at a time period of more than 30, 40,50, 60, 70, 80, 90, 100, or 120 days after being prepared.

The first topical formulation is then added to the skin damage for a 90day time period, for up to 1, 2, 3 or 4 times a day, for up to 30, 60,90 or 120 days or any number of days in between a range defined by anytwo aforementioned values.

The cells in the first formulation are then activated by providing asecond topical formulation to the subject, wherein the providingcomprises instructions to the subject for applying the second topicalformulation over the area of the skin and skin damage that has receivedthe first topical formulation, wherein the second topical formulationactivates the platelet rich plasma, and wherein the activating comprisesadministering the second topical formulation onto the skin of thesubject, and wherein the second topical formulation is applied over theskin that has received the first formulation.

The subject is then monitored by a medical professional. The secondformulation may be sprayed onto the subject or may be applied as alotion.

In an alternative of this example, the first formulation is provided tothe subject and retained at a refrigerated temperature. The firstformulation is then applied to the skin of the subject, such as to theface of the subject, and application of the first formulation increasesthe temperature of the first formulation to room temperature or greater.The change in temperature activates the PRP.

An alternative kit and/or method is provided, wherein the kit providesinstructions for the user to obtain a sample of whole blood from thesubject, and to process the whole blood to obtain PRP. The PRP is thensubmitted to lyophilization to obtain a lyophilized PRP powder. The PRPpowder is weighed and aliquoted in specific amounts in separate vessels,and stored for later usage. When ready for use, the PRP is dissolved byadding to the vessel a dissolution solution, which includes sodiumchloride, phosphate buffered saline, saline, or other dissolving agent,thereby generating a PRP solution. The PRP solution is then added to abase composition as described herein, such that the base compositionpreserves, extends, prolongs, maintains, or sustains the PRP.Furthermore, the base composition preserves, extends, prolongs,maintains, or sustains components of PRP, such as growth factors, orother biomolecules associated with PRP. The base composition preservesthe PRP in the solution for a period of 30, 45, 60, 75, 90, 105, or 120days, or longer, such that the PRP formulation exhibits therapeuticallyeffective results when applied on the skin of the subject over atreatment period. Upon complete usage of the PRP formulation, or after aperiod of time wherein the PRP or the components thereof are no longerviable, a second aliquot of lyophilized PRP powder may be dissolved andcombined with the base composition. Thus, due to the combination of bothprolonged activity of the PRP and components thereof in the basecomposition and the securement of multiple aliquots of lyophilized PRPpowder, following the initial draw of whole blood and subsequentprocessing to obtain PRP, the subject does not need to further drawblood or process the blood to obtain PRP for an extended period of time.

In some embodiments, the lyophilized PRP powder remains stable for aperiod of longer than 22 months. In some embodiments, the PRPformulation in the base composition is applied topically to a subjectfor treatment of post laser or post-operative procedures to enhancinghealing effects, for minimizing scar tissue formation, or for reducingthe appearance of scars.

Example 3: Improved Outcome Based on the Time of Incubation of the FirstFormulation on the Skin of a Subject

In the embodiments herein, subjects were selected based on their skinailments or disorders which may be selected from stretch marks (striae),psoriasis, skin cancer, acne, alopecia, carbuncles, dermatitis, eczema,atopic dermatitis, contact dermatitis, seborrheic dermatitis, cradlecap, perioral dermatitis, shingles, ringworm, melisma, impetigo, acneaestivalis (Mallorca acne), acne conglobate, acne cosmetica (cosmeticacne), acne fulminans (acute febrile ulcerative acne), acne keloidalisnuchae (acne keloidalis, dermatitis papillaris capillitii, folliculitiskeloidalis, folliculitis keloidis nuchae, nuchal keloid acne), adultforehead with scattered red pimples, acne vulgaris, acne mechanica, acnemedicamentosa, acne miliaris necrotica (acne varioliformis), acnevulgaris, acne with facial edema (solid facial edema), blepharophyma,erythrotelangiectatic rosacea (erythematotelangiectatic rosacea,vascular rosacea), excoriated acne (acne excoriee des jeunes filles,Picker's acne), glandular rosacea, gnathophyma, gram-negative rosacea,granulomatous facial dermatitis, rhinophyma, granulomatous perioraldermatitis, halogen acne, hidradenitis suppurativa (acne inversa,pyoderma fistulans significa, Verneuil's disease), idiopathic facialaseptic granuloma, infantile acne, lupoid rosacea (granulomatousrosacea, micropapular tuberculid, rosacea-like tuberculid ofLewandowsky), lupus miliaris disseminatus faciei, metophyma, neonatalacne (acne infantum, acne neonatorum, neonatal cephalic pustulosis),occupational acne, oil acne, ocular rosacea (ophthalmic rosacea,ophthalmorosacea), otophyma, periorificial dermatitis, persistent edemaof rosacea (chronic upper facial erythematous edema, Morbihan's disease,rosaceous lymphedema), phymatous rosacea, pomade acne, papulopustularrosacea (inflammatory rosacea), perifolliculitis capitis abscedens etsuffodiens (dissecting cellulitis of the scalp, dissecting folliculitis,perifolliculitis capitis abscedens et suffodiens of Hoffman), perioraldermatitis, periorbital dermatitis (periocular dermatitis), pyodermafaciale (rosacea fulminans), rhinophyma, rosacea (acne rosacea), rosaceaconglobate, synovitis-acne-pustulosis-hyperostosis-osteomyelitissyndrome (SAPHO syndrome), steroid rosacea, tar acne, skin cancer,tropical acne or a combination thereof.

Whole blood is obtained from a subject having skin damage which is thencentrifuged to separate platelet rich plasma from platelet poor plasmaand red blood cells. The platelet rich plasma (PRP) is then collectedand a topical cosmetic base is added such as a gel, serum, and cream orcombination thereof to form PRP admixed solution thereby forming a firsttopical formulation.

The first topical formulation is then applied to the skin damage for a90 day time period, for up to 1, 2, 3 or 4 times a day, for up to 30,60, 90 or 120 days or any number of days in between a range defined byany two aforementioned values.

Example 4: Study of the Formulation

In a study of 6 patients, the first formulation is applied to subjectswho have skin damage and spots which are due to excessive sun exposure.The patients are selected by a dermatologist for the treatment.

The cells in the first formulation are then activated by providing asecond topical formulation to the subject, wherein the providingcomprises instructions to the subject for applying the second topicalformulation over the area of the skin and skin damage that has receivedthe first topical formulation, wherein the second topical formulationactivates the platelet rich plasma, and wherein the activating comprisesadministering the second topical formulation onto the skin of thesubject, wherein the second topical formulation is applied over the skinthat has received the first formulation.

The subject is then monitored by a medical professional. Two patientsare selected to have the second formulation applied as a lotion, twopatients are selected to have the second formulation applied as a foamand two patients are selected to have the second formulation applied asa spray.

The first formulation is applied on each patient for 30 days. The secondformulation is applied on the area of application of the firstformulation for each patient for 30 days. Alternatively, the secondformulation is applied each day after until 30 days after a singleapplication of the first formulation. The first formulation is washedoff once it is fully activated by the second formulation. The firstformulation can be applied 1, 2, 3, 4 times a day and therefore eachtime the first formulation is applied the second formulation would bethen applied and kept on the face until fully activated.

The patients are monitored for improvement of their skin in the area oftreatment each week for two months after application. Improvement of theskin tone, coloring, skin damage and spots is expected to be markedlyimproved for all patients receiving treatment. However, the greatestimprovement in skin tone, coloring, skin damage and spots is expected inthe patients that received the second formulation as a spray.

Example 5: The Base of Formulation 1

In some embodiments, the first formulation is provided, wherein thefirst formulation comprises a base into which the cells are mixedthereby forming the first formulation. In some embodiments, the basecomposition comprises aqua (water), Lonicera japonica (honeysuckle)flower extract, glycerin, niacinamide, polyacrylate crosspolymer-6,Lonicera caprifolium (honeysuckle) flower extract, Persea gratissima(avocado) oil, Aloe barbadensis leaf juice, sodium gluconate, sodiumhyaluronate (L), Althaea officinalis (marshmallow) root extract,O-cymen-5-OL, sodium hydroxide, glucose (D), sodium chloride, sodiumcitrate, sodium acetate, sodium bicarbonate, tocopheryl acetate(D-alpha), potassium phosphate, potassium chloride, magnesium chloride,phenoxyethanol, Anthemis nobilis (chamomile) flower oil, Rosmarinusofficinalis (rosemary) leaf oil, Thymus vulgaris (thyme) flower/leafoil, benzyl alcohol, caprylyl glycol, caprylhydroxamic acid, orcombinations thereof.

In some embodiments, formulation 1 and/or formulation 2 comprises skinpenetrators, preservatives and/or botanicals. In some embodiments, theskin penetrators include Fulvate fractions (Omni BioceuticalInnovations) liposomes, nanoparticles, dimethylsulfoxide, sodium laurylsulfate, hyaluronic acid, nanostructured lipid.

In some embodiments, the a method of diminishing pore size, diminishingbacteria damage, diminishing red areas, reducing skin damage caused bysun, and maintaining or improving skin tone in a subject in need thereofis provided. The method comprises obtaining whole blood from a subjecthaving an area of skin damage; centrifuging the blood to separateplatelet rich plasma from platelet poor plasma and red blood cells;collecting the platelet rich plasma (PRP); adding a base to the PRP toform PRP admixed solution thereby forming a first topical formulation;providing the first topical formulation to the subject for treatment ofthe area of skin damage for a 90 day time period, wherein the providingcomprises instructions for the subject to topically apply the firsttopical formulation to the area of skin damage 1, 2, 3 or 4 times a day,for up to 30, 60, 90 or 120 days or any number of days in between arange defined by any two aforementioned values; and providing a secondtopical formulation to the subject, wherein the providing comprisesinstructions to the subject for applying the second topical formulationover the area of skin damage that has received the first topicalformulation, and wherein the second topical formulation activates theplatelet rich plasma. In some embodiments, the base comprises botanicalsand preservatives. In some embodiments, the base comprises aqua (water),Lonicera japonica (honeysuckle) flower extract, glycerin, niacinamide,polyacrylate crosspolymer-6, Lonicera caprifolium (honeysuckle) flowerextract, Persea gratissima (avocado) oil, Aloe barbadensis leaf juice,sodium gluconate, sodium hyaluronate (L), Althaea officinalis(marshmallow) root extract, 0-cymen-5-OL, sodium hydroxide, glucose (D),sodium chloride, sodium citrate, sodium acetate, sodium bicarbonate,tocopheryl acetate (D-alpha), potassium phosphate, potassium chloride,magnesium chloride, phenoxyethanol, Anthemis nobilis (chamomile) floweroil, Rosmarinus officinalis (rosemary) leaf oil, Thymus vulgaris (thyme)flower/leaf oil, benzyl alcohol, caprylyl glycol, caprylhydroxamic acid,or combinations thereof.

In some alternatives, the base comprising aqua (water), Lonicerajaponica (honeysuckle) flower extract, glycerin, niacinamide,polyacrylate crosspolymer-6, Lonicera caprifolium (honeysuckle) flowerextract, Persea gratissima (avocado) oil, Aloe barbadensis leaf juice,sodium gluconate, sodium hyaluronate (L), Althaea officinalis(marshmallow) root extract, O-cymen-5-OL, sodium hydroxide, glucose (D),sodium chloride, sodium citrate, sodium acetate, sodium bicarbonate,tocopheryl acetate (D-alpha), potassium phosphate, potassium chloride,magnesium chloride, phenoxyethanol, Anthemis nobilis (chamomile) floweroil, Rosmarinus officinalis (rosemary) leaf oil, Thymus vulgaris (thyme)flower/leaf oil, benzyl alcohol, caprylyl glycol, caprylhydroxamic acid,or combinations thereof, is provided in a kit.

The kit comprises a first formulation having a topical cosmetic base,gel, serum, and cream or combination thereof, wherein the firstformulation is admixed with platelet rich plasma, and wherein the firstformulation further comprises nutrients, botanicals, preservatives orskin penetrators or a combination thereof; and a spray bottle or aerosolcontainer holding a second formulation, the second formulation includinga platelet rich plasma activator in liquid suspension. The secondformulation comprises calcium, magnesium, sodium, components of snakevenom or combinations thereof. In some embodiments, the component ofsnake venom comprises Batroxobin. In some alternatives of the kit, the abase is provided wherein the base comprises aqua (water), Lonicerajaponica (honeysuckle) flower extract, glycerin, niacinamide,polyacrylate crosspolymer-6, Lonicera caprifolium (honeysuckle) flowerextract, Persea gratissima (avocado) oil, Aloe barbadensis leaf juice,sodium gluconate, sodium hyaluronate (L), Althaea officinalis(marshmallow) root extract, O-cymen-5-OL, sodium hydroxide, glucose (D),sodium chloride, sodium citrate, sodium acetate, sodium bicarbonate,tocopheryl acetate (D-alpha), potassium phosphate, potassium chloride,magnesium chloride, phenoxyethanol, Anthemis nobilis (chamomile) floweroil, Rosmarinus officinalis (rosemary) leaf oil, Thymus vulgaris (thyme)flower/leaf oil, benzyl alcohol, caprylyl glycol, caprylhydroxamic acid,or combinations thereof.

Example 6: A Base Composition for the First Formulation

In some embodiments, the first formulation is provided as described inExample 1. The first formulation comprises a base composition in whichthe cells are mixed, thereby forming the first formulation. In someembodiments, a kit is provided, wherein the kit comprises a firstformulation having a topical base wherein PRP is admixed into, therebyforming the first formulation. In some embodiments of the kit, the basecomprises aqua (water), Lonicera japonica (honeysuckle) flower extract,glycerin, niacinamide, polyacrylate crosspolymer-6, Lonicera caprifolium(honeysuckle) flower extract, Persea gratissima (avocado) oil, Aloebarbadensis leaf juice, sodium gluconate, sodium hyaluronate (L),Althaea officinalis (marshmallow) root extract, O-cymen-5-OL, sodiumhydroxide, glucose (D), sodium chloride, sodium citrate, sodium acetate,sodium bicarbonate, tocopheryl acetate (D-alpha), potassium phosphate,potassium chloride, magnesium chloride, phenoxyethanol, Anthemis nobilis(chamomile) flower oil, Rosmarinus officinalis (rosemary) leaf oil,Thymus vulgaris (thyme) flower/leaf oil, benzyl alcohol, caprylylglycol, caprylhydroxamic acid, or combinations thereof.

Example 7: The Activator Spray Comprising the Second Formulation

The second formulation may be provided as an activator spray. Theactivator spray comprises Aqua (Water), Calcium Chloride (Can be anyplatelet activator), Polysorbate 80, Propylene Glycol, Sodium Chloride,Anthemis nobilis (Chamomile) Flower Oil, Rosa canina (Rose Hip) FruitOil, Thymus vulgaris (Thyme) Flower/Leaf Oil, Althaea officinalis(Marshmallow) Root Extract, Hamamelis virginiana (Witch Hazel) Water,Alcohol, Glycerin, Ethylhexylglycerin, Citric Acid, Potassium Sorbate,Phenoxyethanol.

Example 8: Longevity of PRP when Formulated in Base Composition

The following example demonstrates methods of increasing the longevityand activity of PRP over time when combined in a base composition.

PRP was collected from a subject. Five compositions were prepared induplicate as follows: base composition alone; PRP alone; basecomposition+PRP; base composition+PRP+activator spray composition; basecomposition+PRP+calcium chloride. One group of formulations is preparedand stored at room temperature for a period of 0, 5, 15, 30, 45, 60, 75,and 90 days, and the second group of formulations is prepared and storedat 4° C. for a period of 0, 5, 15, 30, 45, 60, 75, and 90 days.

The base composition of Example 1 is combined with PRP, and PRP activityis assessed by ELISA activity and Western blot analysis at time point 0,5, 15, 30, 45, 60, 75, and 90 days to verify that the growth factorsremain active over the time period. Various growth factors are analyzedover time, including EGF, FGF basic, PDGF-AB, TGF-B1, and VEGF. Samplesfor the ELISA and Western blot analysis is performed at room temperatureover the test period or at 4° C. during the test period, and includesthe following conditions: the base composition alone (control), PRPalone (either fresh or lyophilized—control), base composition+PRP, basecomposition+PRP+activator (including one or more of calcium chloride,autologous thrombin, batroxobin, bovine thrombin, or collagen). Theactivator is included either as a direct component added by mixing or asprepared activator composition, such as a spray.

Platelets are analyzed via apoptosis. Platelet apoptosis can betriggered by multiple cell-external chemical stimuli, including plateletagonists thrombin and calcium ionophore A23187, pro-apoptoticanti-platelet antibodies, von Willebrand factor (VWF) in combinationwith the antibiotic ristocetin, mimetics of pro-apoptotic ‘BH3-only’proteins of the Bcl-2 family and other chemical stimuli, as well as byexposure of platelets to very high pathological shear stresses. Thesechemical and physical stimuli induce transformation of resting(non-apoptotic) platelets to an apoptotic state. Depending on the natureof the trigger, this transformation is accompanied by stimulation ofvarious apoptotic events, which may include DWm depolarization, MPTPformation, expression, activation and translocation to mitochondria ofpro-apoptotic members of Bcl-2 family proteins (such as Bax, Bak andBid), cytochrome c release from mitochondria to the cytosol, activationof caspases, cleavage of cytoskeletonproteins, PS exposure on the outerleaflet of plasmamembrane, platelet shrinkage, shedding of plateletderived MIPs, and blebbing, and filopod formation on platelet membrane.Driven by the plasma membrane alterations, damaged platelets can berecognized by the reticuloendothelial system as ‘unwanted’ cells andremoved from the circulation. Different markers of platelet apoptosiscan characterize apoptotic events in different cellular compartments,such as mitochondria, cytosol and plasma membrane, as well as at thewhole-cell level. Various markers, methods, and probes may be used forstudying platelet apoptosis, as shown in Table 2, and as described infurther detail in Gyulkhendanyan et al., BJH, 2013.

TABLE 2 Markers of apoptosis Cell compartments Methods Detecting probesUpstream markers Am depolarization mitochondria Flow cytometry DiOC6(3),JC-1 Bax, Bak and Bcl-2 Cytosol and outer Flow cytometry;Anti-Bax/-Bak/-Bcl- proteins expression and mitochondrial Western blot 2antibodies + FITC- translocationto membrane conjugated mitochondriasecondary antibody; anti-Bax/-Bak/-Bcl- 2 antibodies + HRP- conjugatedsecondary antibody; anti-cytochrome c antibody Cytochrome c releaseMitochondrial Western blot Anti-cytochrome c intermembrane antibodyspace and cytosol Downstream markers Caspase-3 activation cytosol Flowcytometry FAM-DEVD-FMK Phosphatidylserine Plasma membrane Flow cytometryFITC or PE labeled exposure annexin V Microparticle shedding Whole-celllevel Flow cytometry; FSC-anti-GPIIbIIa scanning electron dot plotanalysis microscopy Platelet shrinkage Whole-cell level Flow cytometry;FSC histogram scanning electron analysis microscopy Membrane blebbingPlasma membrane Scanning electron and filopod extrusion microscopy

ELISA, Western blot, or flow cytometry analysis are performed onapoptotic members of Bcl-2 family proteins Bax, Bak, and Bid for timepoints of 0, 5, 15, 30, 45, 60, 75, and 90 days. Expression of Bcl-2family proteins in each sample (control, base composition with PRP, andbase composition with PRP and activator), and is determined by flowcytometry.

In addition, ELISA, Western blot, and flow cytometry analysis isperformed for cytochrome c release, caspase release, cytoskeletonproteins, and platelet derived MPs that are associated with platelets inPRP at time points 0, 5, 15, 30, 45, 60, 75, and 90 days where a gradualincrease is observed over time as more platelets go through apoptosis.In addition, the activation of executioner caspase-3 in each sample isobserved, which is dependent on MPTP formation, and is performed at eachtime point for all samples.

Platelet morphology changes as platelet lose stability and activateunder stress, time, activation, and temperature. Platelet morphology isverified by observing microparticle shedding, platelet shrinkage,membrane blebbing, and filopod extrusion. Experimental techniquesinclude flow cytometry, scanning electron microscopy, specificfluorescent probing analysis, and light scattering characteristicanalysis.

Example 9: Base Formulation with PRP for Treating Skin Disorders

The following example demonstrates methods of treating skin disorders byapplying to a patient PRP provided in a base composition. This exampleexamines the ability of the formulations described herein including PRPin a base composition, wherein the PRP is obtained from the subject towhom it will be applied. This allows the creation of a personalized PRPcream to improve the appearance of the individual from whom the PRP washarvested. However, PRP is a large molecular weight protein that doesnot readily penetrate the skin. This research enhances the PRP deliverythrough the adjunctive use of a skin obstruction device, such aselectroporation, ultrasound, radiofrequency, high intensity focusedultrasound, intense pulsed light (IPL), ablative laser, non-ablativelaser, microdermabrasion, hydradermabrasion, iontophoresis, chemicalpeel, plasma, high velocity air, high velocity aqueous solution, orneedling, or a combination thereof. Skin obstruction is used to createchannels through which the PRP can penetrate into the viable epidermisand dermis to induce an anti-aging effect.

Female and male subjects were enrolled in this single site double blindvehicle controlled split face study to evaluate the effect of aPRP-containing cream on facial photoaging. Subjects completed a 3-daywashout of any facial treatment, including moisturizers and cosmetics,except for lip and eye cosmetics. Subjects were asked to continue theirself-selected cleanser unchanged throughout the 8-week study. Subjectswere randomized to determine which side of the face received aPRP-containing cream and which side received a base control cream.Dermatologist investigator and subject assessments for efficacy andtolerability were conducted on a 5-point ordinal scale separately foreach cheek. Transepidermal water loss (TEWL) was performed from theright and left cheeks and Visia CR photography of the front, right, andleft face were completed prior to any treatment as baselinedocumentation.

The base composition with PRP was prepared using the base compositiondescribed in Example 1, formulated as a cream. Subjects applied onequarter sized amount of the cream twice daily into the freshly washedrandomized half of the face to include the right or left cheeks,forehead, periocular, and perioral areas until the cream absorbs. To theother half of face was applied a quarter sized amount of a control cream(base composition alone without PRP) rubbed into the cheek, forehead,periocular, and perioral area twice daily until the cream absorbs. Thissplit face application was performed twice daily (morning and evening)for the duration of the study. The creams were refrigerated during thestudy period.

Subjects underwent a phlebotomy procedure for harvesting of 50 mL ofblood from which 6-9 mL of PRP was prepared according to directionssupplied by the sponsor. Due to patient compliance, lower quantities ofblood may be obtained, such as an amount of less than 50 mL, such as 5,10, 15, 20, 25, 30, 35, 40, or 45 mL. PRP is prepared, wherein the PRPhas platelet counts that are greater than normal platelet counts (forexample, PRP has platelet counts greater than 150,000-350,000 plateletsper microliter). Following phlebotomy, subjects underwentelectroporation for 5 minutes total to the surface of the cheeks,forehead, periocular area, and perioral area. During this time, thesubjects were educated on how to use the device at home for the durationof the study. The device was used in the evening only for 5 minutes. Thedevice was used prior to the application of the study moisturizers inthe evening only. 3 mL of PRP was added to a cosmetic base composition(outlined in Example 1) formulated as a cream that was applied to onerandomized side of the face twice daily. To the other half of the facewas applied a cosmetic base composition cream alone applied twice daily.Neither the subjects nor the investigator knew the identity of thecreams that were applied to each side of the face. Subjects wereinstructed to return to the research facility at weeks 4 and 8.

Subjects were queried at week 4 and 8 for any adverse events. Compliancediaries were checked along with adequacy of the assigned study creamsupply and proper electroporation device functioning. Diaries werechecked for proper use of the electroporation device to the entire facefor 5 minutes in the evening. Dermatologist investigator and subjectassessments for efficacy and tolerability were conducted on a 5-pointordinal scale separately for each side of the face. TEWL is performedfrom the right and left cheeks and Visia CR photography of the front,right, and left face was completed. TEWL measurements (Dermalab, CortexTechnologies, Hadsund, Denmark) were taken from the right cheek in allsubjects. Measurements occur at baseline, week 4, and week 8. Each sideof the face was assessed separately. Visia CR4.3 photographs were takenof the front, right, and left face. Photographs were taken with thefollowing lighting modes: Standard 1, Standard 2, Standard 3, CrossPolarized, and Parallel Polarized. Assessments occurred at baseline,week 4, and week 8.

The dermatologist and subject each assessed efficacy of the formulationby assessing dryness, lack of tactile smoothness, lack of visualsmoothness, lack of softness, lack of luminosity, lack of radiance, lackof firmness, poor skin texture, fine facial lines, wrinkles, poor skintone, mottled hyperpigmentation, overall appearance. All assessmentswere made on a 5-point ordinal scale (0=none, 1=minimal, 2=mild,3=moderate, 4=severe) at baseline, week 4, and week 8. Each side of theface was assessed separately. The dermatologist and subject eachassessed tolerability by assessing itching, stinging, burning, redness,and swelling. All assessments were made on a 5-point ordinal scale(0=none, 1=minimal, 2=mild, 3=moderate, 4=severe) at baseline, week 4,and week 8. Each side of the face was assessed separately.

A primary efficacy endpoint was a statistically significant improvementin facial photoaging, as assessed by the dermatologist investigator,produced by the PRP+cosmetic base composition cream as compared to thecosmetic base cream only when used in conjunction with homeelectroporation device. A secondary efficacy endpoint was astatistically significant improvement in facial photoaging, as assessedby the subjects, produced by the PRP+cosmetic base cream as compared tothe cosmetic base cream only when used in conjunction with anelectroporation device. A tertiary efficacy endpoint was a statisticallysignificant improvement in skin barrier, as measured by a reduction inTEWL, produced by the PRP+cosmetic base cream as compared to thecosmetic base cream only when used in conjunction with anelectroporation device.

The ordinal investigator and subject nonparametric data were analyzedusing a Mann Whitney paired two-tailed analysis evaluating change frombaseline comparing the active PRP+base cream treated side of the face tothe base cream only treated side of the face. The parametric TEWL datawere analyzed using a Student t test comparing the active PRP treatedside of the face to the control vehicle treated side of the face.Significance was defined at p less than or equal to 0.05.

Subjects in the biopsy study underwent a 2 mm punch biopsy from the leftand right preauricular areas, following anesthesia with 2% lidocaineplus epinephrine. The biopsy sites were closed with one suture. Thebiopsies showed increased collagen and elastin expression over time inportions of the skin having the base composition with PRP applied.Biopsies for qPCR and immunohistochemistry showed upregulation of keyfactors of tissue healing. The specimens were processed, sectioned, andhematoxylin and eosin (H&E) stained. H&E stained 5 μm serial sectionsfrom paraffin blocks and were digitally scanned (NanoZoommer, Hamamatsu,Japan). Length profile measurements of the stratum basale and stratumgranulosum layers of the epidermis were obtained, generating a ratiobetween basale/granulosum layers. Nuclear counting and epithelialthickness measurements were conducted. Quantitative immunohistochemistryanalysis for collagen I and elastin were quantified using a colordeconvolution algorithm from digitally scanned immunohistochemistryslides. Quantitative polymerase chain reaction was conducted analyzingCollagen 1A1 (COL1 A), keratinocyte proline rich protein (KPRP), andmatrix metalloproteinase 1 (MMP1) genes.

Quantitative PCR (qPCR) was performed on biopsy tissue samples that werepreserved in RNA later solution. Fold change in mRNA expression betweentreatment and control for each subject was calculated using thefollowing equation:

Fold Change (FC)=2^(−(ΔΔCT))

An average fold change of 2 represents a significant upregulation ingene expression, and a 0.5 fold change represents a 2 fold decrease(downregulation) in gene expression. Next, the opposite integer of theΔΔCT value was determined.

−ΔΔCT=log₂(FC)

Lastly an average FC was calculated for the specific probe analysisaccording to the following formula:

Average FC=2 ^(x) , where x is the average of the −ΔΔCT values

Ordinal investigator and subject nonparametric data were analyzed usinga Mann Whitney paired two-tailed analysis evaluating change frombaseline comparing the active PRP+base composition treated side of theface to the base composition only treated side of the face. Theparametric TEWL, histology, and immunohistochemistry data were analyzedusing a paired Student's t-test (p≤0.05).

All 20 subjects in the main study and the subjects in the biopsysub-study successfully completed the study. No tolerability issues werenoted by either the blinded investigator or the subjects with either thePRP+base composition or the base composition alone. This was confirmedby the lack of change in TEWL readings from both sides of the face. Inaddition, no statistically significant differences between the PRP+basecomposition treatment or the base composition alone treatment was notedby either the blinded investigator or the subjects after 8 weeks of usein any of the assessed parameters. Both sides of the face demonstratedstatistically significant (p<0.001) improvement for dryness, tactilesmoothness, visual smoothness, softness, luminosity, and radiance at 8weeks. PRP+base composition did demonstrate directional improvement ofradiance, luminosity, smoothness at 4 weeks, increasing at 8 weeksversus the base composition alone.

The histopathology findings demonstrated a qualitative improvement inthe PRP+base composition treated group with a trend of greater rete pegpresence in the PRP+base composition group compared to base compositioncontrols (FIGS. 1A-1B). Immunohistochemistry revealed enhanced Collagentype I expression in the PRP+base composition treatment versus the basecomposition without PRP (FIGS. 2A-2B). These collagen findings werefurther supported by the qPCR data. Three target genes (collagen IA,matrix metalloproteinase 1 gene, and keratinocyte proline rich protein)and a housekeeping gene control was evaluated. Results demonstrated a2.5-fold increase in collagen gene upregulation in the PRP treatmentbase composition vs. control base composition for collagen type I.

The PRP+base composition formulation was evaluated for PRP stability asthe effective duration of blood derived products has always been alimiting factor for topical application (FIG. 3 ). Intact PDGF wasidentified in the 4 degree Celsius constantly refrigerated preservativebase composition 90 days after preparation. This was an importantfinding to allow further development of topical PRP cosmetics. The samecompositions were evaluated at 4 degree Celsius with the addition ofactivator (activator spray or CaCl₂)). The formulation was alsoevaluated at room temperature, as shown in FIG. 4 .

Assessments of skin rejuvenation in the base composition compared toPRP+base composition at 4 weeks is shown in FIG. 5A, and at 8 weeks isshown in FIG. 5B, with the 4 week to 8 week comparison of the PRP+basecomposition shown in FIG. 5C, and the comparison of 4 weeks to 8 weeksof the composition alone shown in FIG. 5D. The overall percentage changeis shown in FIG. 6 . The outcomes were also shown as a function ofefficacy parameter scales on a scale of 0 to 4, with the compositionalone shown in FIG. 7A, and the PRP+base composition shown in FIG. 7B.The 0 to 8 week comparisons are shown in FIGS. 7C and 7D.

The PRP+base composition demonstrated a directional enhanced performanceat 4 weeks, increasing in performance at 8 weeks for radiance,luminosity, firmness, and softness versus the base composition alone.Further, the PRP+base composition demonstrated less epidermal cellcompacting and greater cellular hydration versus the base compositionalone. In addition, rete pegs typically extend into the dermis inyounger individuals helping to solidify the integrity ofdermal-epidermal junction (DEJ), improving skin strength. Withincreasing age, the rete pegs regress into the upper epidermis. ThePRP+base composition demonstrated continued elongation of the rete pegsversus the base composition alone, with retraction of the rete pegs intothe upper regions of the epidermis. Less rete peg connectivity to theDEJ results in the fragile crepey skin observed with aging.Immunohistochemistry results demonstrated higher levels of collagen typeI and qPCR results showed upregulation of collagen mRNA.

Each subject was assessed by Visia CR4.3 photographs, with an initialphotograph taken at the starting point (week 0), and a treatmentphotograph taken during treatment periods (week 8). The photographs werecompared to assess each of a variety of skin assessments, includingdryness, tactile smoothness, visual smoothness, softness, luminosity,radiance, firmness, texture, lines, wrinkles, tone, and pigmentation.The photographs show decreased red areas, decreased wrinkles, decreasedpore size, improved skin tone, fewer spotting, indicating the ability ofthe PRP+base composition to mitigate signs of facial photoaging (FIGS.8-15 depict exemplary images showing improvement in wrinkles, acnescars, skin elasticity, sagging skin, skin dryness, rashes, redness,translucency, fine lines, radiance, skin tone, pigmentation,discoloration, blotchiness, scarring, rough and leathery appearance,freckles, moles, actinic keratosis, wound healing, bruising and tearing,ruddiness, uneven texture, fine lines, age spots, or a combinationthereof after treatment).

The side of the face having the base composition in combination with PRPexhibited a diminished pore size, diminished bacteria damage, diminishedred areas, reduced skin damage caused by the sun, improved texture, andenhanced skin tone. In addition, skin of subjects having laser treatmentalso showed improved healing.

With respect to the use of plural and/or singular terms herein, thosehaving skill in the art can translate from the plural to the singularand/or from the singular to the plural as is appropriate to the contextand/or application. The various singular/plural permutations may beexpressly set forth herein for sake of clarity.

It will be understood by those of skill within the art that, in general,terms used herein, and especially in the appended claims (e.g., bodiesof the appended claims) are generally intended as “open” terms (e.g.,the term “including” should be interpreted as “including but not limitedto,” the term “having” should be interpreted as “having at least,” theterm “includes” should be interpreted as “includes but is not limitedto,” etc.). It will be further understood by those within the art thatif a specific number of an introduced claim recitation is intended, suchan intent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

Any of the features of an embodiment of the first through second aspectsis applicable to all aspects and embodiments identified herein.Moreover, any of the features of an embodiment of the first throughthird aspects is independently combinable, partly or wholly with otherembodiments described herein in any way, e.g., one, two, or three ormore embodiments may be combinable in whole or in part. Further, any ofthe features of an embodiment of the first through third aspects may bemade optional to other aspects or embodiments.

What is claimed is:
 1. A base composition for prolonging, preserving, ormaintaining a biologic, the composition comprising: a cell culturenutrient; a biological buffer; a viscosity modifying agent; and abotanical extract.
 2. The base composition of claim 1, wherein the cellculture nutrient comprises fetal bovine serum (FBS), glucose, humanplatelet lysate, bovine serum albumin, fibroblast growth supplement,vitamins, trace elements, antioxidants, minerals, amniotic cell culturesupplements, or lipo-polysaccharides.
 3. The base composition of claim1, wherein the biological buffer comprises sodium, potassium, magnesium,calcium, alpha hydroxy acid, beta hydroxy acid, polyhydroxy acid,hyaluronic acid, carboxylic acid, or a cell culture buffering agent, ora derivative or any combination thereof, and wherein the biologicalbuffer preserves the biologic at about or above physiological pH.
 4. Thebase composition of claim 1, wherein the viscosity modifying agentcomprises polyacrylate crosspolymer-6.
 5. The base composition of claim1, wherein the botanical extract comprises an aqueous ferment extract,an alcohol extract, a salicylate, a phenolic compound, or aphytonutrient.
 6. The base composition of claim 5, wherein the aqueousferment extract comprises a probiotic fermented botanical extract. 7.The base composition of claim 6, wherein the probiotic comprisesLactobacillus, Lactobacillus casei, Lactobacillus fermentum,Lactobacillus paracasei, Lactobacillus derivatives, Leuconostoc,Leuconostoc derivatives, Bifidobacterium, Bifidobacterium longum,Bifidobacterium derivatives, Streptococcus, Streptococcus thermophilus,Streptococcus derivatives, Saccharomyces ferment filtrate, or Bacillusferment.
 8. The base composition of claim 6, wherein the probioticfermented botanical extract comprises Cocos nucifera fruit fermentedwith Lactobacillus, Leuconostoc kimchi, or Leuconostoc with radish rootferment filtrate.
 9. The base composition of claim 5, wherein thesalicylate comprises an Aspen Bark isolate.
 10. The base composition ofclaim 5, wherein the phenolic compound comprises a thymol.
 11. The basecomposition of claim 10, wherein the thymol comprises a thymol isomer, acresol, or O-cymen-5-ol.
 12. The base composition of claim 5, whereinthe phytonutrient is a Sambucus nigra fruit extract derivative, Populustremuloides bark extract derivative, or Ribes nigrum fruit extractderivative.
 13. The base composition of claim 1, wherein the biologic isplatelet rich plasma (PRP).
 14. The base composition of claim 1, furthercomprising apple extract or apple skin extract.
 15. The base compositionof claim 1, further comprising ethylhexylglycerin, phenoxyethanol, EDTA,polysorbate, butylene glycol, xanthan gum, propanediol, tocopherylacetate, glycerin, shea butter, water, one or more saccharides, or grapeextract.
 16. The base composition of claim 1, wherein the biologicalbuffer is sodium citrate.
 17. The base composition of claim 1, furthercomprising phenoxyethanol and capryl glycol.